Pulmonary administration of chemically modified insulin

ABSTRACT

The present invention provides active, hydrophilic polymer-modified derivatives of insulin. The insulin derivatives of the invention are, in one aspect, suitable for delivery to the lung and exhibit pharmakokinetic and/or pharmacodynamic properties that are significantly improved over native insulin.

This application claims the benefit of priority of U.S. Provisionalpatent application Ser. No. 60/292,423, filed May 21, 2001 the contentof which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to bioactive, hydrophilicpolymer-modified insulin derivatives for delivery to the lung byinhalation. Methods for preparing and administering such derivatives arealso provided.

BACKGROUND OF THE INVENTION

Insulin is a polypeptide hormone that is produced in the pancreaticβ-cells of normal (non-diabetic) individuals. Human insulin is a 51amino acid polypeptide hormone with a molecular weight of about 5800daltons. The insulin molecule is composed of two peptide chains (an Aand a B chain) containing one intrasubunit and two intersubunitdisulfide bonds. The A chain is composed of 21 amino acids while the Bchain is composed of 30 amino acids. The two chains of insulin form ahighly ordered structure with several α-helical regions in both the Aand the B chains. Interestingly, the isolated chains of insulin areinactive. In solution, insulin can exist as a monomer or as a dimer oras a hexamer. Insulin is hexameric in the highly concentratedpreparations used for subcutaneous therapy but becomes monomeric as itis diluted in body fluids. Insulin is necessary for regulatingcarbohydrate metabolism by reducing blood glucose levels; a systemicdeficiency of insulin causes diabetes. The survival of diabetic patientsdepends on the frequent and long-term administration of insulin tomaintain acceptable blood glucose levels.

Current insulin formulations possess deficiencies that can lead toserious medical complications in the treatment of diabetes. Forinstance, the standard zinc insulin preparation most commonly used bydiabetics exists as a suspension of microcrystals of inactive hexamericinsulin. Dissolution of the microcrystals followed by dissociation ofthe hexamer into the active monomer form can lead to delayed andindividually variable absorption of insulin into the bloodstream (F.Liu, et al., Bioconjugate Chem., 8, 664-672 (1997); T. Uchio, et al.,Adv. Drug Del. Rev., 35, 289-306 (1999); K. Hinds, et al., BioconjugateChem., 11, 195-201 (2000). Formulations of insulin also suffer fromphysical instability due to the tendency of insulin to form fibrils andinsoluble precipitates. Precipitation is especially problematic forformulations intended for use in insulin pumps. Formulated insulin isalso prone to chemical degradation, e.g., non-enzymatic deamidation andformation of high molecular weight transformation products such ascovalent insulin dimers (Brange, J., et al., Pharm. Res., 9, 715-726(1992); Brange, J., et al., Pharm. Res., 9, 727-734 (1992). There issignificant evidence that the incidence of immunological responses toinsulin may result from the presence of these covalent aggregates ofinsulin (Robbins, D.C., et al., Diabetes, 36, 838-841 (1987). Moreover,even highly purified human insulin is slightly immunogenic. (Kim, ibid.)

Apart from the formulation instability problems noted above, there arealso numerous drawbacks associated with current insulin therapies froman administration standpoint. Insulin is most commonly administered bysubcutaneous injection, typically into the abdomen or upper thighs.Insulin may also be administered intravenously or intramuscularly. Inorder to maintain acceptable blood glucose levels, it is often necessaryto inject insulin at least once or twice per day, with supplementalinjections of insulin being administered when necessary. Aggressivetreatment of diabetes can require even more frequent injections, wherethe patient closely monitors blood glucose levels using a homediagnostic kit. The administration of insulin by injection isundesirable in a number of respects. First, many patients find itdifficult and burdensome to inject themselves as frequently as necessaryto maintain acceptable blood glucose levels. In fact, many Type 2patients avoid going on insulin for years because of needle phobia. Suchreluctance can lead to non-compliance, which in the most serious casescan be life-threatening. Moreover, systemic absorption of insulin fromsubcutaneous injection is relatively slow, frequently requiring from 45to 90 minutes, even when fast-acting insulin formulations are employed.Thus, it has long been a goal to provide alternative insulinformulations and routes of administration which avoid the need forself-injection and which can provide rapid systemic availability ofinsulin.

Numerous non-injectable formulation types such as oral and nasal havebeen explored, however, no commercially viable oral or nasal-baseddelivery system for insulin has been developed as a result of theseefforts (Patton, et al., Adv. Drug Delivery Reviews, 1, 35 (2-3),235-247 (1999)), mainly due to very low and variable bioavailability(Hilsted, J., et al., Diabetologia 38, 680-684, (1995)). Althoughbioavailability can be increased with absorption enhancers, these agentscan damage the mucosa.

However, inhaleable formulations of insulin have been developed whichappear to be quite promising in overcoming many of the problems notedabove. For example, U.S. Pat. No. 5,997,848 (Patton, et al., InhaleTherapeutic Systems, Inc.) describes dry powder formulations of insulinwhich (i) are chemically and physically stable at room temperature, and(ii) when inhaled, are rapidly absorbed through the epithelial cells ofthe alveolar region into the blood circulation. The rapid-acting insulinformulations and methods described therein avoid the need for burdensomeself-injections, and have been shown in three month human efficacystudies to provide equivalent glucose control in Type I and Type IIinsulin-dependent diabetics when compared to subcutaneous injection(Patton, et al., Adv. Drug Delivery Reviews, 1, 35 (2-3), 235-247(1999)). The dry powder insulin formulations described by Patton, etal., while overcoming the problems of formulation instability andpatient non-compliance, still require frequent (e.g., mealtime)inhalations of insulin for effective control of glucose levels.Moreover, a typical insulin dosing regime of this type, based on rapidacting inhaleable insulin, still requires a single injection oflong-acting insulin at bedtime for Type I and some Type II diabetics.Thus, there still exists a need for active, soluble, stable forms ofinsulin that require less frequent dosing, i.e., long-acting insulinformulations, preferably administrable by inhalation.

Long-acting insulin formulations are ideally characterized as having avery slow onset and a prolonged, relatively flat peak of action. Currentlong acting injectable insulin formulations, e.g., ultralente (extendedinsulin zinc suspension) and protamine zinc insulin suspension, are veryunsatisfactory. These formulations tend to peak rather than provide alow basal concentration of insulin, are unpredictable, and typicallyexhibit a duration of action of no longer than about a day. The longhalf-life of ultralente insulin makes it difficult to determine theoptimal dosage range, and protamine zinc insulin is rarely used becauseof its unpredictable and prolonged course of action (Goodman & Gilman,“The Pharmacological Basis of Therapeutics, Ninth Ed., Hardman andLimbird, eds, 1996, p. 1500). Other long-acting injectable formulationswhich have been explored unsuccessfully include albumin-bound insulinand cobalt-insulin hexamer formulations (Hoffman, A., Ziv E., Clin.Pharmacokinet, 33(4):285-301 (1997)).

A number of long-acting pulmonary insulin formulations have also beenexplored. These include liposomes containing a large excess of lipidrelative to insulin (Liu. F-Y, et al., Pharm. Res. 10, 228-232, (1993)),porous poly(lactic acid-co-glycolic acid) (PLGA) insulin particles(Edwards, D. A., et al., Science 276(5320), 1868-1871 (1997)), nebulizedPLGA nanospheres (Kawashima, Y., et al., J. Controlled Release, 62(1-2):279-287 (1999)) and phospholipid/protamine insulin formulations(Vanbever, R., et al., Proc. Control Rel. Bioact. Mater. 25, 261-262(1998)). Unfortunately, all of these formulations have provenunsatisfactory, due to either low bioavailabilities when administered inrats, or due to formulation insufficiencies. Thus, a long-felt needexists for optimized long-acting insulin formulations that arebioactive, physically and chemically stable, water-soluble, andpreferably monomeric. Ideally, such formulations will preferably besuited for pulmonary administration.

SUMMARY OF THE INVENTION

In one aspect, the present invention is based upon compositions ofinsulin for administration to the systemic circulation via the deeplung. Specifically, the compositions of the invention comprise aconjugate of insulin covalently coupled to one or more molecules of anon-naturally occurring hydrophilic polymer. In a preferred embodiment,the non-naturally occurring, hydrophilic polymer covalently coupled toinsulin is a polyalkylene glycol such as polyethylene glycol (PEG),although all of the embodiments set forth herein may be equally appliedto other non-naturally occurring hydrophilic polymers.

In general, an insulin-polymer conjugate of the invention will exhibitpharmacokinetic and pharmacodynamic properties improved over nativeinsulin, particularly when administered to the lung. In one embodiment,the PEG-insulin conjugates provided herein exhibit good absolutebioavailabilities when administered to the lung and deep lung. In aparticular embodiment, a PEG-insulin conjugate of the invention ischaracterized by an absolute pulmonary bioavailability that is greaterthan that of native insulin. Preferably, a PEG-insulin conjugate of theinvention is characterized by having an absolute pulmonarybioavailability that is at least 1.5-2.0 times greater than that ofnative insulin. In a more preferred embodiment, a PEG-insulin conjugatein accordance with the invention is characterized by an absolutepulmonary bioavailability that is greater than about 15%, even morepreferably greater than about 20% or most preferably greater than about30%.

In yet another embodiment, a PEG-insulin conjugate of the invention,when administered pulmonarily, exhibits a Tmax (time required to reachmaximum concentration) that is at least 1.5 times that of nativeinsulin, or more preferably is at least 2 or 3 times, or even morepreferably that is at least five times that of native insulin.

PEGs for use in the conjugates of the invention may possess severaldifferent features. In one embodiment of the invention, the polyethyleneglycol-portion of a PEG-insulin conjugate as described herein isend-capped with an inert or non-reactive terminal group such as analkoxy group or more specifically methoxy group.

In an alternative embodiment, the polyethylene glycol portion of theconjugate will possess an architecture particularly well suited forattachment to insulin including linear polyethylene glycols andmulti-armed or branched polyethylene glycols. In yet another embodiment,a PEG-insulin conjugate may comprise two mono-functionally-derivatizedinsulin molecules interconnected by a di-activated polyethylene glycol(insulin-PEG-insulin). Alternatively, an insulin molecule within this“dumbell” architecture may be further modified by additional PEGs.

In another embodiment, a PEG-insulin conjugate of the inventioncomprises a forked polyethylene glycol having a branching moiety at oneend of the polymer chain and two free reactive groups (or a multiple oftwo) linked to the branching moiety for covalent attachment to insulin.In this embodiment of the invention, the branched architecture ofpolyethylene glycol allows attachment of the polymer chain to two ormore molecules of insulin.

The polyethylene glycol-portion of an insulin conjugate of the inventionmay optionally contain one or more degradable linkages.

Typically, insulin is covalently coupled to PEG via a linking moietypositioned at a terminus of the PEG. Preferred linking moieties for usein the invention include those suitable for coupling with reactiveinsulin amino groups such as N-hydroxysuccinimide active esters, activecarbonates, aldehydes, and acetals.

In yet another embodiment, a PEG covalently coupled to insulin in aconjugate of the invention will comprise from about 2 to about 300subunits of (OCH₂CH₂), preferably from about 4 to 200 subunits, and morepreferably from about 10 to 100 subunits.

In an alternative embodiment, a PEG covalently coupled to insulin willpossess a nominal average molecular weight of from about 200 to about10,000 daltons. In a preferred embodiment, the PEG will possess anominal average molecular weight from about 200 to about 5000 daltons.In yet a more preferred embodiment, the PEG will possess a nominalaverage molecular weight from about 200 to about 2000 daltons or fromabout 200 to about 1000 daltons.

In a particular embodiment, the insulin portion of the conjugatecomprises native human insulin.

In yet another embodiment, the conjugate of the composition of theinvention possesses a purity of greater than about 90% (i.e., of theconjugate portion of the composition, 90% or more by weight comprisesone or more PEG-insulins). That is to say, compositions of the inventionmay be characterized by a high degree of purity of conjugated insulincomponent, i.e., the composition is absent detectable amounts of freepolyethylene glycol species and other PEG-related impurities.

In one embodiment, a composition of the invention comprises a conjugatewherein insulin is covalently coupled to PEG at one or more of its aminosites. Insulin contained within a composition of the invention may bemono-substitituted (i.e., having only one PEG covalently coupledthereto). Particular mono-substituted PEG-insulin conjugates inaccordance with the invention possess a polyethylene glycol moietycovalently attached to a position on the insulin molecule selected fromthe group consisting of PheB1, GlyA1 and LysB29.

In a preferred embodiment, the PEG moiety is covalently attached at thePheB 1 site of insulin. In a one embodiment, at least about 75% of theB-1Phe sites on insulin are covalently coupled to PEG. In anotherembodiment, at least about 90% of the B-1 Phe sites on insulin arecovalently coupled to PEG.

Compositions of the invention may also comprise a mixture ofmono-conjugated and di-conjugated PEG insulin having any one or more ofthe features described above. Such compositions may further comprise atri-conjugated PEG insulin.

In an alternative embodiment, a PEG insulin conjugate in accordance withthe invention is characterized by a rate of proteolysis that is reducedrelative to non-pegylated or native insulin.

A composition according to the invention may also comprise a mixture ofa PEG-insulin conjugate and non-chemically modified or native insulin.

Also encompassed is a composition as described above in aerosolizedform.

Compositions of the invention may be dissolved or suspended in liquid orin dry form, and may additionally comprise a pharmaceutically acceptableexcipient.

Also provided herein is a bioactive polyethylene glycol-insulinconjugate suitable for administration by inhalation to the deep lung.

In yet another aspect, the invention provides a method for delivering aPEG-insulin conjugate to a mammalian subject in need thereof byadministering by inhalation a PEG-insulin composition as previouslydescribed in aerosolized form.

The invention also provides in another aspect, a method for providing asubstantially non-immunogenic insulin composition for administration tothe lung. The method includes the steps of covalently coupling insulinto one or more molecules of a non-naturally occurring hydrophilicpolymer conjugate as described herein, and administering the compositionto the lung of subject by inhalation, whereby as a result, the insulinpasses through the lung and enters into the blood circulation.

In another aspect, provided is a method for providing a prolonged effectinsulin composition for administration to the lung of a human subject.The method includes covalently coupling insulin to one or more moleculesof a non-naturally occurring hydrophilic polymer to provide acomposition that includes an insulin-hydrophilic polymer conjugate, andadministering the composition to the lung of the subject by inhalation.Upon the administering step, insulin passes through the lung and entersinto the blood circulation and elevated blood levels of insulin aresustained for at least 8 hours post administration.

A PEG-insulin conjugate of the invention, when aerosolized andadministered via inhalation, is useful in the treatment of diabetesmelliltus (DM).

These and other objects and features of the invention will become morefully apparent when the following detailed description is read.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plot of the rate of enzymatic digestion of an illustrativePEG-insulin conjugate (“750-2 PEG insulin”) versus an unmodified insulincontrol as described in detail in Example 6.

FIG. 2 is a plot of mean serum insulin concentrations following i.v.administration of illustrative compositions of pegylated (5K PEGInsulin) versus non-pegylated insulin (details are provided in Example7);

FIG. 3 is a plot of blood glucose concentrations following i.v.administration of exemplary compositions of pegylated (5K PEG Insulin)versus non-pegylated insulin (details are provided in Example 7);

FIG. 4 is a plot of mean serum insulin concentrations followingintratracheal instillation of pegylated (150 μg/animal, 5K PEG Insulin)versus non-pegylated human insulin (40 μg/animal) in male rats (Example8);

FIG. 5. is a plot of mean blood glucose concentrations followingintratracheal instillation of pegylated (150 μg/animal, 5K PEG Insulin)versus non-pegylated human insulin (40 μg/animal) in male rats (Example8);

FIG. 6 is a plot of mean serum insulin concentrations followingintratracheal instillation of pegylated (750-1 PEG Insulin) versusnon-pegylated human insulin in male rats (Example 9);

FIG. 7. is a plot of mean blood glucose concentrations followingintratracheal instillation of pegylated (750-1 PEG Insulin) versusnon-pegylated human insulin in male rats (Example 9);

FIG. 8 is a plot of mean serum insulin concentrations followingintratracheal instillation of pegylated (750-1 PEG Insulin, 80 and 160μg/animal) versus non-pegylated human insulin (80 μg/animal) in malerats (Example 10);

FIG. 9. is a plot of mean blood glucose concentrations followingintratracheal instillation of pegylated (750-1 PEG Insulin, 80 and 160μg/animal) versus non-pegylated human insulin (80 μg/animal) in malerats (Example 10);

FIG. 10 is a plot of mean serum insulin concentrations followingintratracheal instillation of pegylated (750-2 PEG Insulin, 80μg/animal) versus non-pegylated human insulin (80 μg/animal) in malerats (Example 10);

FIG. 11. is a plot of mean blood glucose concentrations followingintratracheal instillation of pegylated (750-1 PEG Insulin, 80μg/animal) versus non-pegylated human insulin (80 μg/animal) in malerats (Example 11);

FIG. 12 is a plot of mean blood glucose concentrations followingintratracheal instillation of pegylated (2K PEG Insulin, 300 μg/animal,600 μg/animal, 900 μg/animal, and 1200 μg/animal) versus non-pegylatedhuman insulin (80 μg/animal) in male rats (Example 12);

FIG. 13 is a plot of mean serum insulin concentrations following i.v.administration of an illustrative composition of pegylated (2K PEGInsulin) versus non-pegylated insulin (details are provided in Example13); and

FIG. 14 is a plot of blood glucose concentrations following i.v.administration of an exemplary composition of pegylated (2K PEG Insulin)versus non-pegylated insulin (details are provided in Example 13).

DETAILED DESCRIPTION OF THE INVENTION

The design, synthesis and characterization of various representativePEG-insulin conjugates have been optimized for pulmonary delivery to thelung. Although the preparation of PEG-insulin molecules has beenpreviously described, the use of covalent coupling of PEG for providingprolonged action formulations of inhaleable insulin has not beenpreviously demonstrated. In this regard, the challenge facing theapplicants was to provide PEG-insulin conjugates having the optimalbalance of number, location, structure, and molecular weight of PEGchains covalently attached to the insulin molecule to provide insulincompositions suitable for administration to the systemic circulation,preferably via the deep lung. Surprisingly, in light of the above, theinventors have discovered certain PEG- modified insulin formulationshaving one or more of the following features: (i) that are bioactive,i.e., that demonstrate at least about 5% of the activity of nativeinsulin, or preferably have a bioactivity that is at least eithersubstantially maintained or only minimally reduced from that of nativeinsulin, or even more preferably, having an activity that is improvedover native insulin, (ii) that are absorbed from the lung into thebloodstream (as opposed to “sticking” in the lung), (iii) that arechemically and physically stable, (iv) that, when administered to thelung, achieve blood levels of insulin that are elevated above baselinefor at least about 8 hours post administration, (v) that are resistantto enzymatic attack by insulin-degrading enzymes, and (vi) that exhibithalf lives that are extended over non-pegylated insulin whenadministered by inhalation, the details of which will become apparentwhen reading the following description.

I. Definitions

The following terms as used herein have the meanings indicated.

As used in the specification, and in the appended claims, the singularforms “a”, “an”, “the”, include plural referents unless the contextclearly dictates otherwise.

“Insulin” as used herein is meant to include proinsulin and encompassesany purified isolated polypeptide having part or all of the primarystructural conformation (that is to say, contiguous series of amino acidresidues) and at least one of the biological properties of naturallyoccurring insulin. In general, the term “insulin” is meant to encompassnatural and synthetically-derived insulin including glycoforms thereofas well as analogs thereof including polypeptides having one or moreamino acid modifications (deletions, insertions, or substitutions) tothe extent that they substantially retain at least 80% or more of thetherapeutic activity associated with full length insulin (prior tochemical modification with a hydrophilic, non-naturally occurringpolymer as described herein). The insulins of the present invention maybe produced by any standard manner including but not limited topancreatic extraction, recombinant expression and in vitro polypeptidesynthesis. Native or wild-type insulin refers to insulin having an aminoacid sequence corresponding to the amino acid sequence of insulin asfound in nature. Native or wild-type insulin can be natural (i.e.,isolated from a natural source) or synthetically produced.

A “physiologically cleavable” or “degradable” bond is a weak bond thatreacts with water (i.e., is hydrolyzed) under physiological conditions.Preferred are bonds that have a hydrolysis half life at pH 8, 25° C. ofless than about 30 minutes. The tendency of a bond to hydrolyze in waterwill depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include butare not limited to carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides andoligonucleotides.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include but are not limited to thefollowing: carbon—carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

“PEG” or polyethylene glycol, as used herein, is meant to encompass anywater-soluble poly(alkylene oxide). Most typically, PEGs for use in thepresent invention will contain the following structure,“—CH₂CH₂O(CH₂CH₂O)_(n)CH₂CH₂—, wherein the terminal groups or actualarchitecture of the overall PEG moiety may vary. One commonly employedPEG is end-capped PEG, wherein one terminus of the PEG is capped with arelatively inactive group, typically an alkoxy group such as methoxy(—OCH₃), while the other terminus is a hydroxyl group that can then besubjected to chemical modification. Specific PEG forms for use inpreparing the insulin conjugates of the invention, such as branched,linear, forked PEGs, and the like, will be described in greater detailbelow.

“PEG-insulin conjugate” refers to an insulin molecule (as previouslydefined) having covalently linked or coupled thereto at least onepolyethylene glycol moiety, and possessing any measurable degree ofinsulin activity (e.g., from about 2% to about 100% or more of thebiological activity of native insulin).

“Nominal average molecular weight” in the context of a hydrophilic,non-naturally occurring polymer of the invention such as PEG, refers tothe mass average molecular weight of polymer, typically determined bysize exclusion chromatography, light scattering or intrinsic velocity in1,2,4-trichlorobenzene. The polymers of the invention are typicallypolydisperse, possessing a low polydispersity value of less than about1.05.

A “lipophilic moiety” is one which, when attached to a hydrophilicpolymer in accordance with the invention, either by a degradable ornon-degradable bond, is effective to substantially alter the hydrophilicnature of the polymer and thus the polymer-insulin conjugate. Typicallipophilic groups such as fatty acids will comprise from about 12-22carbon atoms.

A “substantially non-immunogenic” insulin conjugate of the inventionpossesses a reduced immunogenicity relative to native insulin.Immunogenicity may be assessed by determining antibody titres in mice orpreferably in rabbits upon administration of a PEG insulin conjugaterelative to non-modified insulin.

“Alkyl” refers to hydrocarbon chains, typically ranging about 1 to 15atoms in length. The hydrocarbon chains are preferably but notnecessarily saturated and may optionally contain additional functionalgroups attached thereto. The hydrocarbon chains may be branched orstraight chain. Exemplary alkyl groups include ethyl, propyl,1-methylbutyl, 1-ethylpropyl and 3-methylpentyl. In one preferredembodiment of the invention, conjugates comprising an alkylated PEG, andin particular, a linear alkylated PEG, are those having an alkyl portionthat is not a fatty acid or other lipophilic moiety.

“Lower alkyl” refers to an alkyl group containing from 1 to 5 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl.

“Absolute pulmonary bioavailability” is the percentage of a drug dose(e.g., of a PEG-insulin conjugate in accordance with the invention)delivered to the lungs that is absorbed and enters the blood circulationof a mammal relative to an intravenous dose of native insulin.Representative model systems for determining absolute pulmonarybioavailabilities include rat, dog, rabbit, and monkey. The inhaleablePEG-insulin compositions of the invention are, in one aspect,characterized by an absolute pulmonary bioavailability of at least about20% in plasma or blood, with absolute pulmonary bioavailabilitiesgenerally ranging from about 10% to 30% or more. Generally, dependingupon the specific nature of the PEG-insulin conjugate, a conjugate ofthe invention will possess an absolute pulmonary bioavailability of atleast about one of the following: 10%, 12%, 15%, 18%, 20%, 22%, 25%,30%, 32%, 35% or greater. Absolute pulmonary bioavailability may beestimated by measuring absorption from direct intratrachealadministration, instillation, or by inhalation of a PEG-insulinconjugate composition.

“Distribution phase”, in reference to the half-life of a PEG-insulinconjugate, refers to the initial rapid phase during which insulindisappears from the plasma. The terminal slow or elimination phasehalf-life refers to the slow phase during which insulin is eliminatedfrom the body.

“Prolonged effect” insulin refers to insulin having a duration of effect(i.e., elevated blood levels above baseline) of at least about 6 hours,preferably of at least about 8 hours.

“Glucose levels that are suppressed” refers to blood levels of glucose(e.g., after administration of a PEG-insulin conjugate of the invention)that are suppressed below baseline or basal levels.

“Pharmaceutically acceptable salt” includes but is not limited to aminoacid salts, salts prepared with inorganic acids, such as chloride,sulfate, phosphate, diphosphate, hydrobromide, and nitrate salts, orsalts prepared with an organic acid, such as malate, maleate, fumarate,tartrate, succinate, ethylsuccinate, citrate, acetate, lactate,methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate,salicylate and stearate, as well as estolate, gluceptate andlactobionate salts. Similarly salts containing pharmaceuticallyacceptable cations include, but are not limited to, sodium, potassium,calcium, magnesium, aluminum, lithium, and ammonium (includingsubstituted ammonium).

“Amino acid” refers to any compound containing both an amino group and acarboxylic acid group. Although the amino group most commonly occurs atthe position adjacent to the carboxy function, the amino group may bepositioned at any location within the molecule. The amino acid may alsocontain additional functional groups, such as amino, thio, carboxyl,carboxamide, imidazole, etc. An amino acid may be synthetic or naturallyoccurring, and may be used in either its racemic or optically active(D-, or L-) forms, including various ratios of enantiomers.

“Peptides” are composed of two or more amino acids joined by a peptidebond. Peptides can be homo- or hetero-peptides (i.e., composed ofidentical or different amino acid residues as defined above), and canvary in length from two amino acids to several hundred amino acids

“Dry powder” refers to a powder composition that typically contains lessthan about 10% moisture.

A composition that is “suitable for pulmonary delivery” refers to acomposition that is capable of being aerosolized and inhaled by asubject so that a portion of the aerosolized particles reach the lungsto permit penetration into the lower respiratory tract and alveoli. Sucha composition is considered to be “respirable” or “inhaleable”.

“Aerosolized” particles are liquid or solid particles that are suspendedin a gas, typically as a result of actuation (or firing) of aninhalation device such as a dry powder inhaler, an atomizer, a metereddose inhaler, or a nebulizer.

“Emitted Dose” or “ED” provides an indication of the delivery of a drugformulation from a suitable inhaler device after a firing or dispersionevent. More specifically, for dry powder formulations, the ED is ameasure of the percentage of powder which is drawn out of a unit dosepackage and which exits the mouthpiece of an inhaler device. The ED isdefined as the ratio of the dose delivered by an inhaler device to thenominal dose (i.e., the mass of powder per unit dose placed into asuitable inhaler device prior to firing). The ED is anexperimentally-determined parameter, and is typically determined usingan in-vitro device set up which mimics patient dosing. To determine anED value, a nominal dose of dry powder, typically in unit dose form, isplaced into a suitable dry powder inhaler (such as that described inU.S. Pat. No. 5,785,049, assigned to Inhale Therapeutic Systems) whichis then actuated, dispersing the powder. The resulting aerosol cloud isthen drawn by vacuum from the device, where it is captured on a taredfilter attached to the device mouthpiece. The amount of powder thatreaches the filter constitutes the emitted dose. For example, for a 5 mgdry powder-containing dosage form placed into an inhalation device, ifdispersion of the powder results in the recovery of 4 mg of powder on atared filter as described above, then the emitted dose for the drypowder composition is: 4 mg (delivered dose)/5 mg (nominaldose)×100=80%. For non-homogenous powders, ED values provide anindication of the delivery of drug from an inhaler device after firingrather than of dry powder, and are based on amount of drug rather thanon total powder weight. Similarly for MDI and nebulizer dosage forms,the ED corresponds to the percentage of drug which is drawn from adosage form and which exits the mouthpiece of an inhaler device.

“Fine particle dose” or “FPD” is defined as the mass percent of powderparticles having an aerodynamic diameter less than 3.3 μm, typicallydetermined by measurement in an Andersen cascade impactor. Thisparameter provides an indication of the percent of particles having thegreatest potential to reach the deep lung of a patient for systemicuptake of a drug substance.

A “dispersible” or “dispersive” powder is one having an ED value of atleast about 30%, more preferably 40-50%, and even more preferably atleast about 50-60% or greater.

“Mass median diameter” or “MMD” is a measure of mean particle size,since the powders of the invention are generally polydisperse (i.e.,consist of a range of particle sizes). MMD values as reported herein aredetermined by centrifugal sedimentation, although any number of commonlyemployed techniques can be used for measuring mean particle size (e.g.,electron microscopy, light scattering, laser diffraction).

“Mass median aerodynamic diameter” or “MMAD” is a measure of theaerodynamic size of a dispersed particle. The aerodynamic diameter isused to describe an aerosolized powder in terms of its settlingbehavior, and is the diameter of a unit density sphere having the samesettling velocity, in air, as the particle. The aerodynamic diameterencompasses particle shape, density and physical size of a particle. Asused herein, MMAD refers to the midpoint or median of the aerodynamicparticle size distribution of an aerosolized powder determined bycascade impaction, unless otherwise indicated.

“Pharmaceutically acceptable excipient or carrier” refers to anexcipient that may optionally be included in the compositions of theinvention. Preferred for compositions for inhalation are excipients thatcan be taken into the lungs with no significant adverse toxicologicaleffects to the subject, and particularly to the lungs of the subject.

“Pharmacologically effective amount” or “physiologically effectiveamount” is the amount of a PEG-insulin conjugate present in atherapeutic composition as described herein that is needed to provide adesired level of insulin in the bloodstream to result in a target bloodglucose level. The precise amount will depend upon numerous factors,e.g., the particular PEG-insulin, the delivery device employed, thecomponents and physical characteristics of the therapeutic composition,intended patient population, patient considerations, and the like, andcan readily be determined by one skilled in the art, based upon theinformation provided herein.

II. Hydrophilic, Non-Naturally Occurring Polymer-Insulin Conjugates

Several illustrative PEG-insulin conjugates in accordance with theinvention have been prepared. Although polyethylene glycol is apreferred polymer for use in the conjugates of the invention, otherwater-soluble, hydrophilic, non-naturally occurring polymers may also beemployed. Other polymers suitable for use in the invention includepolyvinylpyrrolidone, polyvinylalcohol, polyacryloylmorpholine,polyoxazoline, and poly(oxyethylated polyols) such as poly(oxyethylatedglycerol), poly(oxyethylated sorbitol), and poly(oxyethylated glucose).Polymers comprising subunits or blocks of subunits selected from theabove-described water-soluble polymers may also be used. Additionally,Co-polymers of polyethylene glycol and polypropylene glycol may beemployed. Polymers of the invention are preferably substantially absentfatty acid groups or other lipophilic moieties.

The following section illustrates that with the careful selection of oneor more PEG moieties, pegylation reagents, insulin pegylation sites,pegylation conditions and subsequent conjugate purification, PEG-insulincompositions with the desired clinical properties (improvedpharmacokinetic and/or pharmacodynamic properties) can be obtained.Specific features of the PEG-insulin conjugates of the invention willnow be provided.

A. Structural Features of the Polymer and the Resulting Conjugate

A PEG-insulin conjugate of the invention will typically comprise one ormore PEG chains each having a molecular weight ranging from about 200 toabout 40,000 daltons, and preferably ranging from about 200 to about10,000 daltons. Preferably, a PEG for use in the invention will possessan average molecular weight falling within one of the following ranges:from about 200 to 10,000 daltons, from about 200 to about 7500 daltons,from about 200 to about 6,000 daltons, from about 200 to about 5,000daltons, from about 200 to about 3000 daltons, from about 200 to about2000 daltons, and from about 200 to about 1000 daltons. Exemplaryconjugates prepared with PEGs having molecular weights of 5,000 daltons,2000 daltons and 750 daltons are provided in Examples 1-4.

Preferred PEG-insulins for administration to the lung will possess a PEGmoiety having a molecular weight less than about 5000 daltons,preferably less than about 2000 daltons, and even less than about 1000daltons. In one particular embodiment of the invention, the PEG-insulinconjugate possesses a PEG moiety having one of the following averagemolecular weights: 200, 300, 400, 500, 600, 750, 1000, 1500, 2000, 2500,3000, 3500, 4000 or 5000. Higher molecular weight PEGs may, in certaininstances, be less preferred due to a potential for loss of activity ofthe insulin molecule or, for pulmonary applications, reduced efficiencyin crossing the lung (Example 8).

While lower molecular weight PEGs may be preferred for increasingbioavailability, high molecular weight PEG chains, e.g., having anaverage molecular weight of 5,000, 10,000, 15,000, 20,000, 25,000,30,000 or 40,000 daltons or greater, although generally found todecrease the bioavailability of native insulin, may be preferred forincreasing half-life, particularly in the case of injectableformulations. That is to say, a significant improvement in thepharmacokinetic parameters, e.g., the area under the curve (AUC), for ahigh molecular weight PEG insulin (relative to native), can more thancompensate for its diminished activity.

In terms of the number of subunits, PEGs for use in the invention willtypically comprise a number of (OCH₂CH₂) subunits falling within one ormore of the following ranges: 2 to about 900 subunits, from about 4 toabout 200 subunits, from about 4 to about 170 subunits, from about 4 toabout 140 subunits, from about 4 to about 100 subunits, from about 10 toabout 100 subunits, from about 4 to about 70 subunits, from about 4 toabout 45 subunits, and from about 4 to about 25 subunits.

A PEG-insulin conjugate of the invention may be mono-substituted (i.e.,that is to say, having a PEG attached to a single reactive insulin site)di-substituted (having PEG moieties attached to two reactive sites,tri-substituted, or even have polymer attachments at more than 3 siteson the insulin molecule. Mono-substituted, di-substituted, andtri-substituted insulin are also referred to herein as PEG monomer,dimer, and trimer, respectively. Multi-substituted insulin (meaninginsulin having PEG moieties covalently attached at 2 or more insulinsites) will typically although not necessarily possess the same PEGmoiety attached to each reactive site. That is to say, PEG-insulincompositions having more than one type of PEG moiety attached to insulinare contemplated. Preferred compositions in accordance with theinvention are those containing predominantly monomer and/or dimerinsulin conjugates. Surprisingly, PEG-insulin compositions that are notsite-specific (comprising a mixture of PEG-insulin species having PEGcovalently coupled to more than one reactive site) have been found topossess pharmacokinetic and pharmacodynamic properties improved overnative insulin, in particular, when administered to the lung (Example11).

With respect to the position of PEG-substitution, the insulin moleculepossesses several sites suitable for pegylation, with amino sitesgenerally but not necessarily being most preferred. Specific insulinamino groups suitable for pegylation include the two N-termini, GlyA1and PheB 1, as well as LysB29. These sites on the insulin molecule arealso referred to herein simply as A1, B 1 and B29, respectively.Electrophilically activated PEGs for use in coupling to reactive aminogroups on insulin include mPEG2-ALD, mPEG-succinimidyl propionate,mPEG-succinimidyl butanoate, mPEG-CM-HBA-NHA, mPEG-benzotriazolecarbonate, mPEG-acetaldehyde diethyl acetal, and the like (ShearwaterCorporation, Huntsville, Ala.).

A composition of the invention may, in one embodiment, containpredominantly (greater than 90%) monosubstituted insulin, e.g., mono-A1insulin, mono-B1 insulin, or mono-B29 insulin. Such compositions maycontain: i) mono-A1 insulin, ii) a mixture of mono-A1 insulin andmono-B1 insulin, or iii) a mixture of mono-A1, mono-B1 and mono-B29insulin. Alternatively, a composition of the invention may containpredominantly di-substituted insulin, e.g., di-A1,B1-insulin, ordi-A1,B29-insulin, or di-B1, B29-insulin, or any of the variouscombinations thereof.

Alternatively, a composition in accordance with the invention maycontain a mixture of various PEG-insulin conjugates (i.e., PEG attachedto any one of a combination of possible attachment sites). Using theamino sites on insulin as an example, a composition of the invention maycontain any one or more of the following PEG-insulin conjugates:monoA1-PEG insulin, mono-B1-insulin, mono-B-29 insulin, di-A1,B1-insulin, di-A1,B29-insulin, di-B1,B29-insulin, ortri-A1,B-1,B29-insulin. In one embodiment, preferred are compositionscontaining predominantly monomers and dimers. Representativecompositions may comprise PEG-insulin conjugates mixtures containing atleast about 75% combined monomer and dimer, at least about 80% combinedmonomer and dimer, or at least about 85 to 90% combined monomer anddimer (e.g., Examples 5 and 6).

PheB 1 is a particularly preferred site for chemical modification byattachment of PEG. In particular, a PEG-insulin conjugate compositionfor use in the present invention may also be characterized in oneembodiment as a composition in which at least about 70% of the B-1 siteson insulin are covalently coupled to PEG, regardless of the overallnumber of PEG-insulin species in the composition (e.g., Table 3A,Example 5). Alternative embodiments include those in which at leastabout 75% of the B-1 sites on insulin are covalently coupled to PEG, orin which at least about 80% of the B-1 sites on insulin are covalentlycoupled to PEG, or in which at least about 90% or the B-1 sites oninsulin are covalently coupled to PEG.

Surprisingly, the inventors have discovered that random mixtures ofPEG-insulin (prepared by random rather than site-directed pegylation),when administered to the lung, result in elevated blood levels ofinsulin that are sustained for at least 6 hours, and more typically forat least 8 hours or greater post-administration. Such mixtures areadvantageous not only due to their beneficial pharmacokinetic andpharmacodynamic properties, but because their synthesis is much simpler(does not require multiple synthetic steps, does not require the use ofprotecting groups, does not require multiple purifications, etc.) thanthe corresponding site-specific approach.

Alternative sites in the native insulin molecule that can be chemicallymodified by covalent attachment of PEG include the 2 C-termini, Arg22B,His10B, His5A, Glu4A, Glu17A, Glu13B, and Glu21B.

In addition to native insulin, non-native insulins having one or moreamino acid substitutions, insertions, or deletions may be utilized suchthat additional sites become available for chemical modification byattachment of one or more PEG moieties. This embodiment of the inventionis particularly useful for introducing additional, customizedpegylation-sites within the insulin molecule, for example, for forming aPEG-insulin having improved resistance to enzymatic degradation. Such anapproach provides greater flexibility in the design of an optimizedinsulin conjugate having the desired balance of activity, stability,solubility, and pharmacological properties. Although mutations can becarried out, i.e., by site specific mutagenesis, at any number ofpositions within the insulin molecule, preferred is an insulin variantin which any one of the first four amino acids in the B-chain isreplaced with a cysteine residue. Such cysteine residues can then bereacted with an activated PEG that is specific for reaction with thiolgroups, e.g., an N-maleimidyl polymer or other derivative, as describedin U.S. Pat. No. 5,739,208 and in International Patent Publication No.WO 01/62827. Exemplary sulfhydryl-selective PEGs for use in thisparticular embodiment of the invention include mPEG-forked maleimide(mPEG(MAL)₂), mPEG2-forked maleimide (mPEG2(MAL)₂), mPEG-maleimide(mPEG-MAL), and mPEG2-maleimide (mPEG2-MAL) (Shearwater Corporation).The structures of these activated PEGS are as follows: mPEG-CONHCH[CH₂CONH(CH₂CH₂O)₂CH₂CH₂-MAL, mPEG2-lysine-NH—CH[CH₂CONH(CH₂CH₂O)₂CH₂CH₂-MAL]₂, mPEG-MAL, and mPEG2-lysine-NH—CH₂CH₂NHC(O)CH₂CH₂MAL, respectively.

Additional mutations to the native insulin sequence may be employed, ifnecessary, to increase the bioactivity of a PEG-insulin conjugate whosebiological activity is somewhat compromised as a result of pegylation.One such mutation is Thr8 to a His8. Additional mutations may be found,for example, in Diabetes Care, 13 (9), (1990), the content of which isherein incorporated by reference.

PEGs for use in the present invention may possess a variety ofstructures: linear, forked, branched, dumbbell, and the like. Typically,PEG is activated with a suitable activating group appropriate forcoupling a desired site or sites on the insulin molecule. An activatedPEG will possess a reactive group at a terminus for reaction withinsulin. The term “linker” as used herein is meant to encompass anactivating group positioned at a PEG terminus for reaction with insulin,and may further include additional (typically inert) atoms positionedbetween the PEG portion of the polymer and the activated group at theterminus, for ease in preparing the activated PEG. The linkers maycontain any of a number of atoms, however, preferred are linkerscontaining methylenes intervening between the PEG backbone and theterminal activating group, e.g., as in mPEG-succinimidyl propionate andmPEG-butanoate. Representative activated PEG derivatives and methods forconjugating these agents to a drug such as insulin are known in the artand further described in Zalipsky, S., et al., “Use of FunctionalizedPoly(Ethylene Glycols) for Modification of Polypeptides” in PolyethyleneGlycol Chemistry: Biotechnical and Biomedical Applications, J. M.Harris, Plenus Press, New York (1992), and in Advanced Drug Reviews,16:157-182 (1995).

In one particular embodiment of the invention, the PEG portion of theconjugate is absent one or more lipophilic groups effective tosignificantly modify the water-soluble nature of the polymer or of thepolymer-insulin conjugate. That is to say, the polymer or non-insulinportion of a conjugate of the invention may contain a group of atomsconsidered to be more lipophilic than hydrophilic (e.g., a carbon chainhaving from about 2 to 8-12 carbon atoms), however, if the presence ofsuch a group or groups is not effective to significantly alter thehydrophilic nature of the polymer or of the conjugate, then such amoiety may be contained in the conjugates of the invention. That is tosay, an insulin conjugate of the invention itself is characterized ashydrophilic, rather than lipophilic or amphiphilic. Typically, thepolymer portion of an insulin conjugate, prior to coupling to insulin,whether or not containing such a lipid-loving group, will possess a highhydrophilic/lipophilic balance (HLB) number. The HLB number is basedupon a weight percentage of each type of group (hydrophilic orlipophilic) in a molecule; values typically range from about 1-40. Apolymer for use in the conjugates of the invention is, on a whole,characterized as hydrophilic, regardless of the presence of one or morelipid-loving substituents. In one embodiment of the invention, thepolymer portion of a polymer-insulin conjugate is characterized by anHLB number of greater than 25 and more preferably greater than 30, oreven more preferably greater than 35. In certain embodiments of theinvention where such a lipophilic moiety may be present, the moiety ispreferably not positioned at a terminus of a PEG chain.

Branched PEGs for use in the conjugates of the invention include thosedescribed in International Patent Publication WO 96/21469, the contentsof which is expressly incorporated herein by reference in its entirety.Generally, branched PEGs can be represented by the formulaR(PEG-OH)_(n), where R represents the central “core” molecule and _(n)represents the number of arms. Branched PEGs have a central core fromwhich extend 2 or more “PEG” arms. In a branched configuration, thebranched polymer core possesses a single reactive site for attachment toinsulin. Branched PEGs for use in the present invention will typicallycomprise fewer than 4 PEG arms, and more preferably, will comprise fewerthan 3 PEG arms. Branched PEGs offer the advantage of having a singlereactive site, coupled with a larger, more dense polymer cloud thantheir linear PEG counterparts. One particular type of branched PEG canbe represented as (MeO-PEG-)_(p)R—X, where p equals 2 or 3, R is acentral core structure such as lysine or glycerol having 2 or 3 PEG armsattached thereto, and X represents any suitable functional group that isor that can be activated for coupling to insulin. One particularlypreferred branched PEG is mPEG2-NHS (Shearwater Corporation, Alabama)having the structure mPEG2-lysine-succinimide.

In yet another branched architecture, “pendant PEG” has reactive groupsfor protein coupling positioned along the PEG backbone rather than atthe end of PEG chains as in the previous example. The reactive groupsextending from the PEG backbone for coupling to insulin may be the sameor different. Pendant PEG structures may be useful but are generallyless preferred, particularly for compositions for inhalation.

Alternatively, the PEG-portion of a PEG-insulin conjugate may possess aforked structure having a branched moiety at one end of the polymerchain and two free reactive groups (or any multiple of 2) linked to thebranched moiety for attachment to insulin. Exemplary forked PEGs aredescribed in International Patent Publication No. WO 99/45964, thecontent of which is expressly incorporated herein by reference. Theforked polyethylene glycol may optionally include an alkyl or “R” groupat the opposing end of the polymer chain. More specifically, a forkedPEG-insulin conjugate in accordance with the invention has the formula:R-PEG-L(Y-insulin)_(n), where R is alkyl, L is a hydrolytically stablebranch point and Y is a linking group that provides chemical linkage ofthe forked polymer to insulin, and n is a multiple of 2. L may representa single “core” group, such as “—CH—”, or may comprise a longer chain ofatoms. Exemplary L groups include lysine, glycerol, pentaerythritol, orsorbitol. Typically, the particular branch atom within the branchingmoiety is carbon.

In one particular embodiment of the invention, the linkage of the forkedPEG to the insulin molecule, (Y), is hydrolytically stable. In apreferred embodiment, n is 2. Suitable Y moieties, prior to conjugationwith a reactive site on insulin, include but are not limited to activeesters, active carbonates, aldehydes, isocyanates, isothiocyanates,epoxides, alcohols, maleimides, vinylsulfones, hydrazides,dithiopyridines, and iodacetamides. Selection of a suitable activatinggroup will depend upon the intended site of attachment on the insulinmolecule and can be readily determined by one of skill in the art. Thecorresponding Y group in the resulting PEG-insulin conjugate is thatwhich results from reaction of the activated forked polymer with asuitable reactive site on insulin. The specific identity of such thefinal linkage will be apparent to one skilled in the art. For example,if the reactive forked PEG contains an activated ester, such as asuccinimide or maleimide ester, conjugation via an amine site on insulinwill result in formation of the corresponding amide linkage. Theseparticular forked polymers are particularly attractive since theyprovide conjugates having a molar ratio of insulin to PEG of 2:1 orgreater. Such conjugates may be less likely to block the insulinreceptor site, while still providing the flexibility in design toprotect the insulin against enzymatic degradation, e.g., by insulindegrading enzyme.

In a related embodiment, a forked PEG-insulin conjugate of the inventionis represented by the formula: R-[PEG-L(Y-insulin)₂]_(n). In thisinstance R represents a central core structure having attached theretoat least one PEG-di-insulin conjugate. Specifically, preferred forkedpolymers in accordance with this aspect of the invention are those weren is selected from the group consisting of 1,2,3,4,5,and 6. Exemplarycore R structures may also be derived from lysine, glycerol,pentaerythritol, or sorbitol.

In an alternative embodiment, in any of the representative structuresprovided herein, the chemical linkage between insulin and the polymerbranch point may be degradable (i.e., hydrolytically unstable).Alternatively, one or more degradable linkages may be contained in thepolymer backbone to allow generation in vivo of a PEG-insulin conjugatehaving a smaller PEG chain than in the initially administered conjugate.Such optional features of the polymer conjugate may provide foradditional control over the final desired pharmacological properties ofthe conjugate upon administration. For example, a large and relativelyinert conjugate (i.e., having one or more high molecular weight PEGchains attached thereto, e.g., one or more PEG chains having a molecularweight greater than about 10,000, wherein the conjugate possessesessentially no bioactivity) may be administered, which then either inthe lung or in the bloodstream, is hydrolyzed to generate a bioactiveconjugate possessing a portion of the originally present PEG chain. Inthis way, the properties of the PEG-insulin conjugate may be somewhatmore effectively tailored. For instance, absorption of the initialpolymer conjugate may be slow upon initial administration, which ispreferably but not necessarily by inhalation. Upon in-vivo cleavage ofthe hydrolytically degradable linkage, either free insulin (dependingupon the position of the degradable linkage) or insulin having a smallpolyethylene tag attached thereto, is then released and more readilyabsorbed through the lung and/or circulated in the blood.

In one feature of this embodiment of the invention, the intactpolymer-conjugate, prior to hydrolysis, is minimally degraded uponadministration, such that hydrolysis of the cleavable bond is effectiveto govern the slow rate of release of active insulin into thebloodstream, as opposed to enzymatic degradation of insulin prior to itsrelease into the systemic circulation.

Appropriate physiologically cleavable linkages include but are notlimited to ester, carbonate ester, carbamate, sulfate, phosphate,acyloxyalkyl ether, acetal, and ketal. Such conjugates should possess aphysiologically cleavable bond that is stable upon storage and uponadministration. For instance, a PEG-cleavable linkage-insulin conjugateshould maintain its integrity upon manufacturing of the finalpharmaceutical composition, upon dissolution in an appropriate deliveryvehicle, if employed, and upon administration irrespective of route.

More particularly, as described generally above, PEG-insulin conjugateshaving biodegradable linkages and useful in the present invention arerepresented by the following structures: PEG1-W-PEG2-insulin (where PEG1and PEG2 can be the same or different) or PEG-W-insulin wherein Wrepresents a weak, biodegradable linkage. These conjugates contain PEGarms or portions of PEG arms that are removable (i.e., cleavable)in-vivo. These particular modified insulins are typically substantiallybiologically inactive when intact, either due to the size of the intactPEG-portion of the molecule or due to steric blockage of the activesites on the insulin molecule by the PEG chain. However, such conjugatesare cleaved under physiological conditions to thereby release insulin ora biologically active PEG-insulin capable of absorption into thesystemic circulation, e.g., from the lung. In a first exemplarystructure, the PEG1 portion may possess any of a number of differentarchitectures discussed herein, and will typically possess a molecularweight of at least about 10,000, such that the conjugate is not rapidlyabsorbed upon administration. The PEG2 portion of the moleculepreferably possesses a molecular weight of less than about 5000 daltons,more preferably less than 2000 daltons, and even more preferably lessthan 1000 daltons. Referring now to the secondary exemplary structure,PEG-W-insulin, the PEG portion will generally possess a molecular weightof at least about 10,000 Daltons or more.

In yet another specific embodiment of the invention, the PEG-insulinconjugate has a dumbbell-like structure in which two insulin moietiesare interconnected by a central PEG. More specifically, such conjugatesmay be represented by the structure insulin-Y-PEG-Z-insulin, where Y andZ are hyrolytically stable linking groups linking insulin to the PEGmoiety. In a particular embodiment, Z is an activated sulfone, whichprior to conjugation, is suitable for reaction with thiol groups oninsulin (e.g., cysteines). Alternatively, Y and Z may be any groupsuitable for covalent coupling with insulin. Additional examples areprovided in U.S. Pat. No. 5,900,461, the content of which is expresslyincorporated herein by reference.

Additional representative mono-and di-functional PEGs having eitherlinear or branched structures for use in preparing the conjugates of theinvention may be purchased from Shearwater Corporation (Alabama).Illustrative structures are described in Shearwater's 2001 catalogueentitled “Polyethylene Glycol and Derivatives for BiomedicalApplications”, the contents of which is expressly incorporated herein byreference.

B. Preparation

The reaction conditions for coupling PEG to insulin will vary dependingupon the particular PEG derivative employed, the site of attachment oninsulin and the particular type of reactive group (i.e., lysine versuscysteine), the desired degree of pegylation, and the like, and canreadily be determined by one skilled in the art.

As exemplified in greater detail below, synthesis of the conjugates ofthe invention may be site-directed (Examples 1, 2 and 4) or may berandom (Example 3). Suitable PEG activating groups for reaction withinsulin amine groups (e.g., GlyA1, PheB1, Lys29B), are tresylate,aldehyde, epoxide, carbonylimidazole, active carbonates (e.g.succinimidyl carbonate), acetal, and active esters such asN-hydroxylsuccinimide (NHS) and NHS-derivatized PEGs . Of these, themost reactive are PEG carboxymethyl-NHS, norleucine-NHS, andsuccinimidyl carbonate. Additional PEG reagents for coupling to insulininclude PEG succinimidyl succinate and propionate. PEG active esterssuitable for use in the invention, e.g., having a single propanoic orbutanoic acid moiety, are described in U.S. Pat. No. 5,672,662, thecontents of which is incorporated herein in its entirety. Specificactive esters for use in preparing the conjugates of the inventioninclude mPEG-succinimidyl propionate and mPEG-succinimidyl butanoate(Examples 1-4).

Optimized experimental conditions for a specific conjugate can readilybe determined, typically by routine experimentation, by one skilled inthe art.

Reactive groups suitable for activating a PEG-polymer for attachment toa thiol (sulfhydryl) group on insulin include vinylsulfones,iodoacetamide, maleimide, and dithio-orthopyridine. Particularlypreferred reagents include PEG vinylsulfones and PEG-maleimide.Additional representative vinylsulfones for use in the present inventionare described in U.S. Pat. No. 5,739,208, the content of which isexpressly incorporated herein by reference.

In some instances, the compositions of the invention compriseselectively PEGylated insulin, i.e., the resulting conjugates areessentially homogeneous with respect to the position and degree ofpegylation. That is to say, site selective or site directed pegylationof an amino group will result in an insulin conjugate compositionwherein primarily the intended target position, e.g., PheB1, has a PEGmoiety attached thereto. Depending upon the intended site of pegylation,a protection/deprotection synthetic strategy may be necessary to preventpegylation of non-target reactive sites within the insulin molecule,e.g., by employing a protecting group such as t-BOC(tert-butoxycarbonyl) or di-BOC (di- butoxycarbonyl). Other suitableamino protecting groups include carbobenzoxy (CBZ), trityl derivativessuch as trityl (Tr), dimethoxytrityl (DMTr) and the like. Otherprotecting groups, such as cyclic diacyl groups or nitrophenylsulfenyl(Nps) may also prove useful for protecting amino functions. An exemplarysite-directed synthesis of a 5K-PEG-insulin composition is provided inExamples 1 and 2.

Such site directed coupling chemistry employed to provide the insulinconjugates of the invention results in compositions having a largedegree of substitution at a particular reactive site on the insulinmolecule. These compositions can then, if desired, be further purifiedto provide compositions of essentially pure mono- or di-functionalPEG-insulins.

An essentially pure PEG-insulin composition refers to one comprising aPEG-insulin conjugate that is at least about 90% pure, and preferably atleast about 95% pure by any one of the following analytical methods. Inthis respect, purity refers to PEG-insulin conjugate content. That is tosay, a PEG-insulin conjugate that is at least about 90% pure contains atleast about 90% by weight of PEG-insulin conjugate species, while theother nearly 10% represents impurities that are not PEG-insulinconjugate. PEG-insulin conjugates of the invention are typicallypurified using one or more purification techniques such as ion exchangechromatography, size exclusion chromatography, affinity chromatography,hydrophobic interaction chromatography, and reverse phasechromatography. The overall homogeneity of the resulting PEG-insulin(number of insulin-PEG forms present) can be assessed using one or moreof the following methods: chromatography, electrophoresis, massspectrometry, and in particular, MALDI-MS, and NMR spectroscopy. Oneparticularly useful method for identifying the sites of insulinmodification is RP-HPLC peptide mapping, coupled with a USP identitytest for human insulin using endoproteinase Glu-C (Example 6).

C. Characteristics of PEG-insulin Conjugates

In accordance with one aspect of the invention, provided are PEG-insulinconjugate compositions that are suitable for pulmonary administration.As can be seen by the in-vivo data in Examples 7-11, the PEG insulinconjugates of the invention, when administered to the lung, possesspharmacokinetic and pharmodynamic properties improved over nativeinsulin. It has been shown that insulin can be modified with PEGs havinga molecular weight of up to 5,000K to 10,000 K or greater, and stillmaintain activity. Activity of a representative PEG-insulin conjugate,5K-PEG-insulin, is demonstrated in Example 7. Additionally, as can beseen from the examples provided herein, exemplary PEG-insulin conjugatespossessing PEG chains with average molecular weights ranging from 750daltons, to 2,000 daltons, to 5,000 daltons, when administered bothintravenously and to the lung, are bioactive, are not substantially heldup within the lung when administered to the lung, as evidenced bydetectable serum levels of insulin, and are effective in producing asubstantial suppression of glucose (Examples 7 to 11), which, in certaincases, is over a duration of time significantly greater than thatobserved for native insulin. Moreover, provided herein are PEG-insulinconjugates, which when administered to the lung, exhibit a rapid onsetof action (within 1 hour of administration). A summary ofpharmacokinetic and pharmacodynamic parameters for exemplary PEG-insulincompositions of the invention is provided in Table 13.

In general, a PEG-insulin composition of the invention will possess oneor more of the following characteristics. The PEG-insulin conjugates ofthe invention maintain at least a measurable degree of specificactivity. That is to say, a PEG-insulin conjugate in accordance with theinvention will possesses anywhere from about 2% to about 100% or more ofthe specific activity of native insulin. In one preferred embodiment ofthe invention, the PEG-insulin conjugate will possess at least 10% ormore of the biological activity of unmodified, native insulin and issubstantially non-immunogenic. Preferably, the bioactivity of aconjugate of the invention will range from about 5% to at least about20% or more of the bioactivity of native insulin. The bioactivity of aconjugate of the invention may be characterized indirectly, e.g., bymonitoring blood glucose and insulin levels to generate thecorresponding pharmacodynamic and/or pharmacokinetic data, or by RIA(radioimmunoassay).

In considering serum concentrations of insulin following administrationof a PEG-insulin conjugate, e.g., to the lung, the conjugates describedherein will typically peak (i.e., reach Cmax or the highest point in theconcentration curve) at from around 2 to 8 hours post dose, and moretypically will peak at around 3 to 6 hours or so. Moreover, thechemically modified insulins of the invention, and in particular, theprolonged effect insulin formulations provided herein, are effective inproviding both a measurable glucose-lowering effect and sustainedconcentrations of insulin over a longer period of time than nativeinsulin. More specifically, a PEG-insulin conjugate when administered tothe lung will exhibit elevated levels of insulin (elevated over basal orbaseline levels) for at least about 6 hours and preferably for at least8 hours post administration. Preferably, a PEG-insulin conjugate whenadministered to the lung, results in elevated blood levels of insulinover a prolonged period of at least 9 hours, 10 hours, 12 hours or atleast 14 hours post administration wherein above-basal levels of insulinconjugate are detectable in the bloodstream for such an extendedduration post dose. Representative compositions demonstrating thesefeatures are provided in the Examples.

As described previously, an insulin conjugate of the invention iseffective to lower blood glucose levels. Turning now to the ability ofthe compositions of the invention to suppress blood glucose, a PEGinsulin conjugate when administered, e.g., to the lung, is effective tosuppress blood glucose levels below basal levels for at least 6 hourspost-administration. More particularly, a PEG-insulin composition of theinvention is effective to suppress blood glucose levels below baselinefor at least 8 hours, preferably for at least 10 hours, or morepreferably for at least 12 hours or more post administration.

Moreover, the PEG-insulin formulations of the invention exhibit absolutepulmonary bioavailabilities that are improved over native insulin.Specifically, a PEG-insulin formulation as provided herein possesses anabsolute pulmonary bioavailability that is at least about 1.2 times thatof native insulin, preferably at least about 1.5 times that of nativeinsulin, more preferably is at least about 2 times greater or even morepreferably is at least about 2.5 or 3 times greater than that of nativeinsulin. (Illustrative results are provided in Table 13).

III. Formulations

The polymer-insulin conjugate compositions of the invention may beadministered neat or in therapeutic/pharmaceutical compositionscontaining additional excipients, solvents, stabilizers, etc., dependingupon the particular mode of admistration and dosage form. The presentconjugates may be administered parenterally as well as non-parenterally.Specific administration routes include oral, rectal, buccal, topical,nasal, ophthalmic, subcutaneous, intramuscular, intraveneous,transdermal, and pulmonary. Most preferred are parenteral and pulmonaryroutes.

Pharmaceutical formulations for mammalian and preferably humanadministration will typically comprise at least one PEG-insulinconjugate of the invention together with one or more pharmaceuticallyacceptable carriers, as will be described in greater detail below,particularly for pulmonary compositions. Formulations of the presentinvention, e.g., for parenteral administration, are most typicallyliquid solutions or suspensions, while inhaleable formulations forpulmonary administration are generally liquids or powders, with powderformulations being generally preferred. Additional albeit less preferredcompositions of the chemically modified insulins of the inventioninclude syrups, creams, ointments, tablets, and the like.

Formulations and corresponding doses of insulin will vary with theconcentration bioactivity of the insulin employed. Injectable insulin ismeasured in USP Insulin Units and USP Insulin Human Units (U); one unitof insulin is equal to the amount required to reduce the concentrationof blood glucose in a fasting rabbit to 45 mg/dl (2.5 mM). Typicalconcentrations of insulin preparations for injection range from 30-100Units/mL which is about 3.6 mg of insulin per mL. The amount of insulinrequired to achieve the desired physiological effect in a patient willvary not only with the particulars of the patient and his disease (e.g.,type I vs. type II diabetes) but also with the strength and particulartype of insulin used. For instance, dosage ranges for regular insulin(rapid acting) are from about 2 to 0.3 U insulin per kilogram of bodyweight per day. Compositions of the invention are, in one aspect,effective to achieve in patients undergoing therapy a fasting bloodglucose concentration between about 90 and 140 mg/dl and a postprandialvalue below about 250 mg/dl. Precise dosages can be determined by oneskilled in the art when coupled with the pharmacodynamics andpharmacokinetics of the precise insulin-conjugate employed for aparticular route of administration, and can readily be adjusted inresponse to periodic glucose monitoring.

Individual dosages (on a per inhalation basis) for inhaleableinsulin-conjugate formulations are typically in the range of from about0.5 mg to 15 mg insulin-conjugate, where the desired overall dosage istypically achieved in from about 1-10 breaths, and preferably in fromabout 1 to 4 breaths. On average, the overall dose of PEG-insulinadministered by inhalation per dosing session will range from about 10Uto about 400U, with each individual dosage or unit dosage form(corresponding to a single inhalation) containing from about 5U to 400U.

A. Inhaleable Formulations of Chemically Modified Insulin

As stated above, one preferred route of administration for the insulinconjugates of the invention is by inhalation to the lung. Particularformulation components, characteristics and delivery devices will now bemore fully described.

The amount of insulin conjugate in the formulation will be that amountnecessary to deliver a therapeutically effective amount of insulin perunit dose to achieve at least one of the therapeutic effects of nativeinsulin, i.e., the ability to control blood glucose levels to nearnormoglycemia. In practice, this will vary widely depending upon theparticular insulin conjugate, its activity, the severity of the diabeticcondition to be treated, the patient population, the stability of theformulation, and the like. The composition will generally containanywhere from about 1% by weight to about 99% by weight PEG-insulin,typically from about 2% to about 95% by weight conjugate, and moretypically from about 5% to 85% by weight conjugate, and will also dependupon the relative amounts of excipients/additives contained in thecomposition. More specifically, the composition will typically containat least about one of the following percentages of PEG-insulin: 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more by weight. Preferably,powder compositions will contain at least about 60%, e.g., from about60-100% by weight PEG-insulin. It is to be understood that more than oneinsulin may be incorporated into the formulations described herein andthat the use of the term “agent” or “insulin” in no way excludes the useof two or more insulins or a combination of insulin with another activeagent. (For example, an illustrative PEG-insulin formulation may alsocomprise native insulin).

A.1. Excipients

Compositions of the invention will, in most instances, include one ormore excipients. Preferred are carbohydrate excipients, either alone orin combination with other excipients or additives. Representativecarbohydrates for use in the compositions of the invention includesugars, derivatized sugars such as alditols, aldonic acids, esterifiedsugars, and sugar polymers. Exemplary carbohydrate excipients suitablefor use in the invention include, for example, monosaccharides such asfructose, maltose, galactose, glucose, D-mannose, sorbose, and the like;disaccharides, such as lactose, sucrose, trehalose, cellobiose, and thelike; polysaccharides, such as raffinose, melezitose, maltodextrins,dextrans, starches, and the like; and alditols, such as mannitol,xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), pyranosylsorbitol, myoinositol and the like. Preferred are non-reducing sugars,sugars that can form a substantially dry amorphous or glassy phase whencombined with an insulin conjugate, and sugars possessing relativelyhigh Tgs (e.g., Tgs greater than 40° C., preferably greater than 50° C.,more preferably greater than 60° C., and even more preferably greaterthan 70° C., and most preferably having Tgs of 80° C. and above).

Additional excipients include amino acids, peptides and particularlyoligomers comprising 2-9 amino acids, and more preferably 2-5 mers, andpolypeptides, all of which may be homo or hetero species. Representativeamino acids include glycine (gly), alanine (ala), valine (val), leucine(leu), isoleucine (ile), methionine (met), proline (pro), phenylalanine(phe), trytophan (trp), serine (ser), threonine (thr), cysteine (cys),tyrosine (tyr), asparagine (asp), glutamic acid (glu), lysine (lys),arginine (arg), histidine (his), norleucine (nor), and modified formsthereof. One particularly preferred amino acid is leucine.

Also preferred for use as excipients in inhaleable compositions are di-and tripeptides containing two or more leucyl residues, as described inInhale Therapeutic System's International patent applicationPCT/US00/09785, incorporated herein by reference in its entirety.

Also preferred are di- and tripeptides having a glass transitiontemperature greater than about 40° C., more preferably greater than 50°C., even more preferably greater than 60° C., and most preferablygreater than 70° C.

Although less preferred due to their limited solubility in water,additional stability and aerosol performance-enhancing peptides for usein the invention are 4-mers and 5-mers containing any combination ofamino acids as described above. More preferably, the 4-mer or 5-mer willcomprise two or more leucine residues. The leucine residues may occupyany position within the peptide, while the remaining (i.e., non-leucyl)amino acids positions are occupied by any amino acid as described above,provided that the resulting 4-mer or 5-mer has a solubility in water ofat least about 1 mg/ml. Preferably, the non-leucyl amino acids in a4-mer or 5-mer are hydrophilic amino acids such as lysine, to therebyincrease the solubility of the peptide in water.

Polyamino acids, and in particular, those comprising any of the hereindescribed amino acids, are also suitable for use as stabilizers.Preferred are polyamino acids such as poly-lysine, poly-glutamic acid,and poly(lys, ala).

Additional excipients and additives useful in the present compositionsand methods include but are not limited to proteins, non-biologicalpolymers, and biological polymers, which may be present singly or incombination. Suitable excipients are those provided in InhaleTherapeutic Systems' International Publication Nos. WO 96/32096 and98/16205. Preferred are excipients having glass transition temperatures(Tg), above about 35° C., preferably above about 40° C., more preferablyabove 45° C., most preferably above about 55° C.

Exemplary protein excipients include albumins such as human serumalbumin (HSA), recombinant human albumin (rHA), gelatin, casein,hemoglobin, and the like. The compositions may also include a buffer ora pH adjusting agent, typically but not necessarily a salt prepared froman organic acid or base. Representative buffers include organic acidsalts of citric acid, ascorbic acid, gluconic acid, carbonic acid,tartaric acid, succinic acid, acetic acid, or phthalic acid. Othersuitable buffers include Tris, tromethamine hydrochloride, borate,glycerol phosphate and phosphate. Amino acids such as glycine are alsosuitable.

The compositions of the invention may also include additional polymericexcipients/additives, e.g., polyvinylpyrrolidones, derivatizedcelluloses such as hydroxymethylcellulose, hydroxyethylcellulose, andhydroxypropylmethylcellulose, Ficolls (a polymeric sugar),hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin),polyethylene glycols, and pectin.

The compositions may further include flavoring agents, taste-maskingagents, inorganic salts (e.g., sodium chloride), antimicrobial agents(e.g., benzalkonium chloride), sweeteners, antioxidants, antistaticagents, surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN80”, and pluronics such as F68 and F88, available from BASF), sorbitanesters, lipids (e.g., phospholipids such as lecithin and otherphosphatidylcholines, phosphatidylethanolamines, although preferably notin liposomal form), fatty acids and fatty esters, steroids (e.g.,cholesterol), and chelating agents (e.g., EDTA, zinc and other suchsuitable cations). The use of certain di-substitutedphosphatidylcholines for producing perforated microstructures (i.e.,hollow, porous microspheres) is described in greater detail below. Otherpharmaceutical excipients and/or additives suitable for use in thecompositions according to the invention are listed in “Remington: TheScience & Practice of Pharmacy”, 19^(th) ed., Williams & Williams,(1995), and in the “Physician's Desk Reference”, 52^(nd) ed., MedicalEconomics, Montvale, N.J. (1998).

In one embodiment, a composition in accordance with the invention may beabsent penetration enhancers, which can cause irritation and are toxicat the high levels often necessary to provide substantial enhancement ofabsorption. Specific enhancers, which may be absent from thecompositions of the invention, are the detergent-like enhancers such asdeoxycholate, laureth-9, DDPC, glycocholate, and the fusidates. Certainenhancers, however, such as those that protect insulin from enzymedegradation, e.g., protease and peptidase inhibitors such as alpha-iantiprotease, captropril, thiorphan, and the HIV protease inhibitors,may, in certain embodiments of the invention, be incorporated in thePEG-insulin formulations of the invention. In yet another embodiment,the PEG-insulin conjugates of the invention may be absent liposomes,lipid matrices, and encapsulating agents.

Generally, the pharmaceutical compositions of the invention will containfrom about 1% to about 99% by weight excipient, preferably from about5%-98% by weight excipient, more preferably from about 15-95% by weightexcipient. Even more preferably, the spray dried composition willcontain from about 0-50% by weight excipient, more preferably from 0-40%by weight excipient. In general, a high insulin concentration is desiredin the final pharmaceutical composition. Typically, the optimal amountof excipient/additive is determined experimentally, i.e., by preparingcompositions containing varying amounts of excipients (ranging from lowto high), examining the chemical and physical stability of thePEG-insulin, MMADs and dispersibilities of the pharmaceuticalcompositions, and then further exploring the range at which optimalaerosol performance is attained with no significant adverse effect uponinsulin stability.

A.2. Preparing Dry Powders

Dry powder formulations of the invention comprising a PEG-insulinconjugate may be prepared by any of a number of drying techniques, andpreferably by spray drying. Spray drying of the formulations is carriedout, for example, as described generally in the “Spray Drying Handbook”,5^(th) ed., K. Masters, John Wiley & Sons, Inc., NY, N.Y. (1991), and inPlatz, R., et al., International Patent Publication Nos. WO 97/41833(1997) and WO 96/32149 (1996), the contents of which are incorporatedherein by reference.

Solutions of PEG-insulin conjugates are spray dried in a conventionalspray drier, such as those available from commercial suppliers such asNiro A/S (Denmark), Buchi (Switzerland) and the like, resulting in adispersible, dry powder. Optimal conditions for spray drying thePEG-insulin solutions will vary depending upon the formulationcomponents, and are generally determined experimentally. The gas used tospray dry the material is typically air, although inert gases such asnitrogen or argon are also suitable. Moreover, the temperature of boththe inlet and outlet of the gas used to dry the sprayed material is suchthat it does not cause degradation of the PEG-insulin in the sprayedmaterial. Such temperatures are typically determined experimentally,although generally, the inlet temperature will range from about 50° C.to about 200° C., while the outlet temperature will range from about 30°C. to about 150° C. Preferred parameters include atomization pressuresranging from about 20-150 psi, and preferably from about 30-40 to 100psi. Typically the atomization pressure employed will be one of thefollowing (psi): 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or above.

Respirable PEG-insulin compositions having the features described hereinmay also be produced by drying certain formulation components whichresult in formation of a perforated microstructure powder as describedin WO 99/16419, the entire contents of which are incorporated byreference herein. The perforated microstructure powders typicallycomprise spray-dried, hollow microspheres having a relatively thinporous wall defining a large internal void. The perforatedmicrostructure powders may be dispersed in a selected suspension media(such as a non-aqueous and/or fluorinated blowing agent) to providestabilized dispersions prior to drying. The use of relatively lowdensity perforated (or porous) microstructures or microparticulatessignificantly reduces attractive forces between the particles, therebylowering the shear forces, increasing the flowability and dispersibilityof the resulting powders, and reducing the degradation by flocculation,sedimentation or creaming of the stabilized dispersions thereof.

Alternatively, a PEG-insulin composition for pulmonary delivery maycomprise aerodynamically light particles as described in U.S. Pat. No.6,136,295.

A powdered formulation of the invention may also be prepared bylyophilization, vacuum drying, spray freeze drying, super critical fluidprocessing (e.g., as described in Hanna, et al., U.S. Pat. No.6,063,138), air drying, or other forms of evaporative drying.

In yet another approach, dry powders may be prepared by agglomeratingthe powder components, sieving the materials to obtain agglomerates,spheronizing to provide a more spherical agglomerate, and sizing toobtain a uniformly-sized product, as described, e.g., and in Ahlneck,C., et al., International PCT Publication No. WO 95/09616, 1995,incorporated herein by reference.

Dry powders may also be prepared by blending, grinding, sieving or jetmilling formulation components in dry powder form.

Once formed, the dry powder compositions are preferably maintained underdry (i.e., relatively low humidity) conditions during manufacture,processing, and storage. Irrespective of the drying process employed,the process will preferably result in inhaleable, highly dispersibleparticles comprising a chemically modified insulin as described herein.

A.3. Features of Dry Powder Formulations

Powders of the invention are further characterized by several features,most notably, one or more of the following: (i) consistently highdispersibilities, which are maintained, even upon storage (ii) smallaerodynamic particles sizes (MMADs), (iii) improved fine particle dosevalues, i.e., powders having a higher percentage of particles sized lessthan 3.3 microns MMAD, all of which contribute to the improved abilityof the powder to penetrate to the tissues of the lower respiratory tract(i.e., the alveoli) for delivery to the systemic circulation. Thesephysical characteristics of the inhaleable powders of the invention, tobe described more fully below, are important in maximizing theefficiency of aerosolized delivery of such powders to the deep lung.

Dry powders of the invention are composed of aerosolizable particleseffective to penetrate into the lungs. The particles of the inventionhave a mass median diameter (MMD) of less than about 20-30 μm, or lessthan 20 μm, or less than about 10 μm, preferably less than about 7.5 μm,and more preferably less than about 4 μm, and even less than about 3.5μm, and usually are in the range of 0.1 μm to 5 μm in diameter.Preferred powders are composed of particles having an MMD from about 0.2to 4.0 μm. In some cases, the powder will also contain non-respirablecarrier particles such as lactose, where the non-respirable particlesare typically greater than about 40 microns in size.

The powders of the invention are further characterized by an aerosolparticle size distribution less than about 10 μm mass median aerodynamicdiameter (MMAD), preferably having MMADs less than about 5 μm, morepreferably less than 4.0 μm, even more preferably less than 3.5 μm, andmost preferably less than 3 μm. The mass median aerodynamic diameters ofthe powders will characteristically range from about 0.1-10 μm,preferably from about 0.2-5.0 μm MMAD, more preferably from about1.0-4.0 μm MMAD, and even more preferably from about 1.5 to 3.0 μm.Small aerodynamic diameters can generally be achieved by a combinationof optimized spray drying conditions and choice and concentration ofexcipients.

The PEG-insulin powders of the invention may further be characterized bytheir densities. A powdered composition for inhalation will generallypossess a bulk density from about 0.1 to 10 g/cubic centimeter,preferably from about 0.1-2 g/cubic centimeter, and more preferably fromabout 0.15-1.5 g/cubic centimeter.

The powders will generally have a moisture content below about 20% byweight, usually below about 10% by weight, and preferably below about 5%by weight. Preferred powders in accordance with the invention having amoisture content that is below about one or more of the following weightpercentages: 15%, 10%, 7%, 5%, or 3%. Such low moisture-containingsolids tend to exhibit a greater stability upon packaging and storage.

Additionally, the spray drying methods and stabilizers described hereinare effective to provide highly dispersible PEG-insulin formulations.For powder formulations, the emitted dose (ED) of these powders istypically greater than 30%, and usually greater than 40%. Morepreferably, the ED of the powders of the invention is greater than 50%,and is often greater than 60%.

The compositions described herein also possess good stability withrespect to both chemical stability and physical stability, i.e., aerosolperformance over time. Generally, with respect to chemical stability,the PEG-insulin conjugate contained in the formulation will degrade byno more than about 15% upon spray drying. That is to say, the powderwill possess at least about 85% intact PEG-insulin conjugate, preferablyat least about 90 or 95% intact conjugate, and even more preferably willcontain at least about 97% or greater intact PEG-insulin. Preferably,the spray drying process will result in powders having less than about10% total protein aggregates, that is to say, greater than 90% by weightof the chemically modified insulin being in monomeric form.

With respect to aerosol performance, compositions of the invention aregenerally characterized by a drop in emitted dose of no more than about20%, preferably no more than about 15%, and more preferably by no morethan about 10%, when stored under ambient conditions for a period ofthree months.

A.4. Administration of the Composition

PEG-insulin formulations as described herein may be delivered using anysuitable dry powder inhaler (DPI), i.e., an inhaler device that utilizesthe patient's inhaled breath as a vehicle to transport the dry powderdrug to the lungs. Preferred are Inhale Therapeutic Systems' dry powderinhalation devices as described in Patton, J. S., et al., U.S. Pat. No.5,458,135, Oct. 17, 1995; Smith, A. E., et al., U.S. Pat. No. 5,740,794,Apr. 21, 1998; and in Smith, A. E., et. al., U.S. Pat. No. 5,785,049,Jul. 28, 1998, herein incorporated by reference. When administered usinga device of this type, the powdered medicament is contained in areceptacle having a puncturable lid or other access surface, preferablya blister package or cartridge, where the receptacle may contain asingle dosage unit or multiple dosage units. Convenient methods forfilling large numbers of cavities (i.e., unit dose packages) withmetered doses of dry powder medicament are described, e.g., in Parks, D.J., et al., International Patent Publication WO 97/41031, Nov. 6, 1997,incorporated herein by reference.

Other dry powder dispersion devices for pulmonary administration of drypowders include those described, for example, in Newell, R. E., et al,European Patent No. EP 129985, Sep. 7, 1988; in Hodson, P. D., et al.,European Patent No. EP472598, Jul. 3, 1996; in Cocozza, S., et al.,European Patent No. EP 467172, Apr. 6, 1994, and in Lloyd, L. J. et al.,U.S. Pat. No. 5,522,385, Jun. 4, 1996, incorporated herein by reference.Also suitable for delivering PEG-insulin dry powders are inhalationdevices such as the Astra-Draco “TURBUHALER”. This type of device isdescribed in detail in Virtanen, R., U.S. Pat. No. 4,668,218, May 26,1987; in Wetterlin, K., et al., U.S. Pat. No. 4,667,668, May 26, 1987;and in Wetterlin, K., et al., U.S. Pat. No. 4,805,811, Feb. 21, 1989,all of which are incorporated herein by reference. Other suitabledevices include dry powder inhalers such as Rotahaler® (Glaxo), Discus®(Glaxo), Spiros™ inhaler (Dura Pharmaceuticals), and the Spinhaler®(Fisons). Also suitable are devices which employ the use of a piston toprovide air for either entraining powdered medicament, liftingmedicament from a carrier screen by passing air through the screen, ormixing air with powder medicament in a mixing chamber with subsequentintroduction of the powder to the patient through the mouthpiece of thedevice, such as described in Mulhauser, P., et al, U.S. Pat. No.5,388,572, Sep. 30, 1997, incorporated herein by reference.

An inhaleable PEG-insulin composition may also be delivered using apressurized, metered dose inhaler (MDI), e.g., the Ventolin® metereddose inhaler, containing a solution or suspension of drug in apharmaceutically inert liquid propellant, e.g., a chlorofluorocarbon orfluorocarbon, as described in Laube, et al., U.S. Pat. No. 5,320,094,Jun. 14, 1994, and in Rubsamen, R. M., et al, U.S. Pat. No. 5,672,581(1994), both incorporated herein by reference.

Alternatively, the PEG-insulins described herein may be dissolved orsuspended in a solvent, e.g., water or saline, and administered bynebulization. Nebulizers for delivering an aerosolized solution includethe AERx™ (Aradigm), the Ultravent® (Mallinkrodt), the Pari LC Plus™ orthe Pari LC Star™ (Pari GmbH, Germany), the DeVilbiss Pulmo-Aide, andthe Acorn II® (Marquest Medical Products).

As previously described, the PEG-insulin conjugates described herein canalso be administered parenterally by intravenous injection, or lesspreferably by intramuscular or by subcutaneous injection. Precisecomponents of such formulations can be readily determined by one skilledin the art. Suitable formulation types for parenteral administrationinclude ready-for-injection solutions, dry powders for combination witha solvent prior to use, suspensions ready for injection, dry insolublecompositions for combination with a vehicle prior to use, emulsions andliquid concentrates for dilution prior to administration. For instance,an injectable solution of a PEG-insulin composition of the invention mayinclude the composition dissolved in an aqueous vehicle such as aqueoussodium chloride, Ringers solution, a dextrose-injection solution,lactated Ringers solution and the like, and may include one or morepharmaceutically acceptable compatible excipients or additives asdescribed above.

IV. Utility

The compositions of the invention are useful, when administered by anysuitable route of administration, and preferably by inhalation or byinjection, in a therapeutically effective amount to a mammalian subject,for treating diabetes mellitus, and in particular, type I or type IIdiabetes.

All articles, books, patents and other publications referenced hereinare hereby incorporated by reference in their entirety.

The following examples illustrate, but in no way are intended to limitthe scope of the present invention.

EXAMPLES

Materials and Methods

Polyethylene glycol reagents were obtained from Shearwater Corporation(Huntsville, Ala.).

Human insulin was obtained from Diosynth, Inc.

Example 1 Synthesis of Di-N^(αA1),N^(εB29)-t-Boc-Insulin

A composition composed primarily of mono-pegylated insulin was preparedin a site-specific fashion as set forth in Examples 1 and 2 using anexemplary linear 5,000 Dalton polyethylene glycol.

Di-protected insulin was first prepared as follows. 602 mg of humaninsulin (0.103 mmol) was dissolved in 3.0 mL of anhydrous dimethylsulfoxide (DMSO) containing 166 uL of triethylamine. 50□1 ofdi-tert-butyldicarbonate (0.215 mmol) was added to the insulin solution.After 60 min at room temperature, the reaction solution was poured into240 mL of acetone followed by addition of 3 drops of 6 M HCl to initiateflocculation. The precipitate was isolated by filtration, and dried invacuo. The reaction product was purified by preparative HPLC using aWaters 25×100 mm C18 column (mean particle size, 15 μm; pore size,100A). Mixtures of acetonitrile and 0.1%TFA in deionized water were usedas eluents at a rate of 3.0 mL/min. The product was collected, distilledto remove acetonitrile, and then lyophilized. Yield was 164.8 mg (26.7%,MW ˜6000 by MALDI).

Example 2 Synthesis of Mono-Pegylated mPEG-5K-SPA-PheB1-InsulinConjugate N^(αBI)-Methoxypoly(ethylene glycol)5K-insulin(mPEG5K-PheB1-insulin)

150 mg (˜0.025 mmol) of newly purified di-N^(αA1),N^(εB29)-t-boc-Insulin from Example 1 was dissolved in 4 mL of DMSOcontaining 95 μL of triethylamine. 169 mg (0.032 mmol) of mPEG-SPA-5000(mPEG-succinimidyl propionate, mPEG-O-CH ₂CH₂C(O)O-succinimide, MW5,000) was added to the insulin solution. After incubation overnight (29hours) at room temperature, the resulting mPEG-insulin derivative wasdiluted to 100 mL with D.I. water, dialyzed against D.I. water for 4hours and then lyophilized. The lyophilized product was re-dissolved in4 mL of anhydrous TFA and maintained under N₂ at 0° C. for 1.5 hours toremove the Boc protecting groups. The deprotected mPEG-insulin wasdiluted to 50 mL with D.I. water and dialyzed against 0.1% NH₄HCO₃ andD.I. water overnight. Lyophilization of the product yielded a whitepowder. Yield was 117.6 mg (41.6%, MW˜11311.6 by MALDI).

The percentage of mono-conjugated insulin, based upon mass spectraldata, was approximately 90%, confirming the site specific nature of thissynthetic approach. Additional characterization data is provided inExample 5. Insulin content of the resulting product was 51.3%. For easeof reference, N^(αB1)-Methoxypoly(ethylene glycol)5K-propionamido-insulin or mPEG5K-PheB1-insulin will be referred to hereinas “5K PEG insulin”.

Example 3 Synthesis of mPEG-2K-SPA-Insulin Conjugate

The following approach was utilized to prepare insulin pegylated in anon-site specific (i.e., random) fashion utilizing an exemplary linearpolyethylene glycol having a molecular weight of approximately 2,000Daltons. 0.1012 g of insulin (MW 5826 Da, 0.01737 mmol) was dissolved in0.5 mL of anhydrous DMSO and treated with 50 μL of triethylamine (0.3587mmol, 20 fold molar excess). To the above reaction mixture was added 52mg of m-SPA-2000 (mPEG-succinimidyl propionate, Shearwater Corporation,MW˜2000 Da, 0.02605 mmol, 1.5 fold molar excess). The mixture wasstirred for about 17 hours at room temperature under nitrogen. Thereaction mixture was then dissolved in 0.1% TFA to a total volume of 5.5mL and purified by reverse phase IHPLC using a C-18 column, andacetonitrile/0.1%TFA as eluent). Reverse phase HPLC revealed a mixtureof both mono (one PEG attached) and di-pegylated (two PEGs attached)product; the composition is referred to herein as “2K PEG insulin”.

Yield: 68 mg

Insulin content by RP-HPLC: 50.5 mg

Example 4 Synthesis of mPEG-750 Da -SPA-Insulin Conjugate

A composition composed predominantly of insulin pegylated at the B1 sitewas prepared in a site-specific fashion using a representativepolyethylene glycol modifier, i.e, a linear 750 Dalton polyethyleneglycol having a succinimidyl propionate terminus suitable for covalentattachment to insulin.

4A. Synthesis of Di-N^(A1),N^(B29)-t-Boc-Insulin

Di-protected insulin was prepared as follows. 602 mg of human insulin(0.103 mmol) was dissolved in 3.0 mL of anhydrous dimethyl sulfoxide(DMSO) containing 166 uL of triethylamine. 50ul ofdi-tert-butyldicarbonate (0.215 mmol) was added to the insulin solution.After 60 min at room temperature, the reaction solution was poured into240 mL of acetone followed by addition of 3 drops of 6 M HCl to initiateflocculation. The precipitate was isolated by filtration and dried invacuo. The reaction product was purified by preparative HPLC using aWaters 25×100 mm C18 column (mean particle size, 15um; pore size, 100A).Mixtures of acetonitrile and 0.1%TFA in deionized water were used aseluents at a rate of 3.0 mL/min. The product was collected, distilled toremove acetonitrile, and then lyophilized. Yield was 164.8 mg (26.7%,MW˜6000 by MALDI).

4B. Synthesis of mPEG-750 Da-SPA-PheB1-Insulin Conjugate

63.4 mg (˜0.01056 mmol) of newly purifieddi-N^(A1),N^(B29)-t-boc-Insulin from Example 4A was dissolved in 0.5 mLof DMSO containing 200 uL of triethylamine. 33 mg (0.03173 mmol, MW ofmSPA750 is about 1040Da) of mPEG-SPA-750 (mPEG-succinimidyl propionate,mPEG—O—CH2CH2C(O)O-succinimide, PEG MW 750) was added to the insulinsolution. After incubation overnight (29 hours) at room temperature, 300uL of TFA was added to the reaction mixture and the resultingmPEG-insulin derivative was precipitated in 100 mL ethyl ether and driedunder vacuum. Yield was about 28.5 mg with 21.3 mg of insulin contentmeasured by reverse phase HPLC (33.6%, MW˜6639.3Da by MALDI. Forsimplicity, the composition is referred to herein as “750 PEG Insulin”.

Two different syntheses were carried out on this material both utilizingthe above synthetic methodology with one exception: one synthesis wascarried out at a molar ratio of mPEG-SPA-750 to insulin of 7:1 while theother synthesis was conducted at a molar ratio of mPEG-SPA-750 toinsulin of 3:1. The product compositions resulting from these twopreparations are referred to herein as “750-1 PEG insulin” (molar ratioof PEG reagent to insulin was 7:1) and “750-2 PEG insulin” (molar ratioof PEG reagent to insulin was 3:1).

Example 5 Characterization of Exemplary PEG-Insulin Compositions

The pegylated insulin conjugate compositions described above werefurther characterized by various analytical techniques.

Mass spectrometry was utilized to provide an estimate of the relativeamounts of mono, di, and tri-conjugated insulin (also referred to as PEGinsulin monomer, dimer, and trimer) present in each of the compositionsbased upon relative peak areas. The results are provided in Table 1below.

TABLE 1 Relative Amounts of Mono, Di, and Tri-Conjugated Insulin Basedon Mass Spectrometry PEG-Insulin Composition % Monoconjugate %Diconjugate % Triconjugate 5K PEG insulin 91  4 not determined 750-1 PEG46 39 15 insulin 750-2 PEG 60 32  8 insulin 2K PEG insulin 51 45 notdetermined

Size exclusion chromatography (SEC) was carried out on the 750-1, 750-2and 2K PEG-insulin compositions described above employing two Shodex SECcolumns (part number KW-802.5) assembled in series on a Waters 2690 HPLCsystem. The mobile phase consisted of 22% glacial acetic acid and 33%acetonitrile (V/V) in water. The chromatography data was used as analternative approach for determining the relative amounts of mono, di,and tri-conjugated insulin in each of these compositions. The resultsare presented in Table 2 below. As can be seen by a comparison of thedata in Tables 1 and 2, the two different methods provide results thatare in close agreement with respect to the relative amounts of each typeof conjugate present in the compositions.

TABLE 2 Relative Amounts of Mono, Di, and Tri-Conjugated Insulin Basedon HP-SEC Insulin % Type % Monoconjugate % Diconjugate % TriconjugateOther PEG 750-1 48 47 5 0 PEG 750-2 66 26 7 2 PEG 2000 40 51 nd 9

Additional studies were carried out to determine the distribution of thevarious positional conjugates in each of three exemplary compositions,i.e., the extent of substitution at each of the three possibleattachment sites, A-1Gly, B-1Phe or B-29 Lys. Dithiothreitol (DTT,Sigma) was used to reduce the disulfide bonds in the insulin samples,causing the covalent attachments between the insulin A and B chains tobe broken.

To carry out the reduction reactions, the PEG-insulin samples weredissolved in 8 M urea containing 0.4 M ammonium bicarbonate at about 0.2mg/mL of equivalent insulin mass for each conjugated species. DTT wasdissolved in water (7 mg/ml) to form an aqueous DTT solution. One partof DTT solution was then added to 5 parts of each of the insulinsolutions, and the reduction reaction was carried out at 50° C. for 15minutes. The reduced 750 PEG insulin compositions were alkylated withiodoacetamide (Sigma). Six parts of the PEG-insulin solutions werereacted with 1 part of 100 mM iodoacetamide prior to chromatography andenzyme digestion. The reaction products were then analyzed by HPLC.Percent conjugation to either the A or B chain of insulin was estimatedbased upon the amount of insulin A or B chain that eluted later than thecontrol (and attributed to conjugation to polyethylene glycol). Theselate-eluting peaks were therefore missing at the expected retentiontimes for the control. Relative peak areas were used to provide anindication of the percent conjugation of polyethylene glycol to eitherthe A or B chain of insulin.

To further explore the relative amount of PEG attached to B-29 Lysversus B1-Phe, the reduced and alkylated A and B chains of the 750-1 and750-2 PEG insulin compositions from the DTT-reductions described abovewere further digested with the sequencing grade enzyme EndoproteinaseGlu-C (Sigma). A solution containing the enzyme at 0.125 μg/μL inaqueous ammonium bicarbonate was prepared. Prior to enzyme solutionaddition, the insulin concentration in each of the reduced reactionmixtures was 0.05 μg/μL in 8 M urea containing 0.4 M ammoniumbicarbonate. 1 part enzyme solution was then added to 40 parts insulinsolution. Digestion with Endoproteinase Glu-C enzyme produces theinsulin peptide fragments of A1-A4, A5-A17, A18-A21, B1-B13, B14-B21,AND B22-B30.

Fragments resulting from the enzymatic digests for both the A and Bchains of the 750-1 and 750-2 PEG insulin compositions were analyzed byHPLC to estimate the overall distribution of PEG attachment sites toinsulin for each of these compositions. The percent of the peak missingrelative to the control provided an estimation of the amount of thefragment conjugated to PEG, since that fragment eluted elsewhere on thechromatogram.

TABLE 3A Distribution of PEG Attachment Sites for Exemplary PEG-InsulinFormulations % of A1 Sites % of B-1 sites % of B-29 Sites Insulin typeConjugated Conjugated Conjugated PEG 750-1 30 95 21 PEG 750-2 11 95 15PEG 2000 63 85* *no digestion data, reduced recovery only

The numbers in Table 3A are based on the possibility of each site being100 percent conjugated. For example, each mono-species has 3 possibleconfigurations (mono-A1, mono-B1, mono-B-29) and each diconjugate has 3configurations (di-A1, B-1; di-A-1,B-29; and di-B-1, B-29). Looking atthe data in Table 3A, e.g., for PEG-750-1, over all of the possiblespecies present in the composition, 95% of the PEG-insulin conjugatespossess a polyethylene glycol covalently attached at the B-1 site.

TABLE 3B Various Conjugate Species Possible TRI- Types MONO-conjugateDI-conjugate conjugate HMWP Species # 1 2 3 4 5 6 7 8 Point of A-1 B-1B-29 (A-1 + (A-1 + (B-1 + (A-1 + Conjugated Conjugation B-1) B-29) B-29)B-1 + Insulin B-29) Dimers

Example 6 Comparison of the Rate of Enzymatic Digestion of 750-2 PEGInsulin Versus Unmodified Insulin

The rate of enzymatic digestion of 750-2 PEG-insulin by chymotrypsin wascompared to that of insulin.

An insulin control and a PEG 750 insulin-2 composition was prepared at 1mg/mL in phosphate buffered saline solution at pH 7.8. Chymostrypsin wasprepared at 1 mg/mL in 1 mM HCl solution. 1 part of the enzyme solutionwas added to 20 parts of the insulin solution. Small aliquots of themixed solution were withdrawn approximately every hour.

A RP(reverse phase)-HPLC method was developed using a C-18 column with amobile phase containing sodium perchlorate, phosphoric acid, andacetonitrile. An acetonitrile gradient was employed to elute theassortment of PEGylated insulin species in a group of slightly resolvedpeaks monitored at 214 nm. The group of peaks were integrated manuallyas one peak and labeled as PEG insulin. As the digestion proceeded, theloss of intact PEG insulin and insulin were plotted (FIG. 1). Theirhalf-life in the presence of chymotrypsin was estimated.

The length of time required for enzymatic digestion of half of theconcentration of the main component of the 750-2 PEG insulin compositionwas five times greater than for unmodified insulin. That is to say, ittook five times longer for digestion by chymostrypsin of half theconcentration of the illustrative pegylated insulin than for regularinsulin. These results demonstrate the potential of PEG insulinconjugates for prolonged residence time in the alveoli due to enhancedresistance to proteolytic degradation when compared to unmodifiedinsulin.

Example 7 Evaluation of Serum Glucose and Insulin ConcentrationsFollowing Intraveneous Administration of 5K PEG Insulin in Rats(P-2001-015)

This study was conducted to determine whether the activity of insulin inthe 5K PEG insulin composition was retained upon chemical modificationwith an exemplary 5K polyethylene glycol chain, and to explore the doseand glucose response curves for these compositions when administeredintraveneously.

Pre-cannulated (jugular/femoral vein [JVC/FVC]) male Sprague Dawley Rats(325-350 grams) having an access port threaded under the skinexternalizing at the nape of the neck were supplied by Hilltop LabAnimals Inc. (P.O. Box 183, Scottdale, Pa. 15683). The jugular cannulaswere filled with a solution (lumen filler) of pharmaceutical gradePolyviny-Pyrrolidone (PVP—MW 40,000), physiological saline and sodiumheparin to retain patency. The nylon filament plug sealing the cannulawas removed and replaced with a Monoject blunt cannula 23G×1 (VWR#53498-484) on the day of the study. The test system included 1 male ratrandomly chosen for the placebo group, 2 male rats randomly chosen forthe non-pegylated group, and 4 male rats randomly chosen for thePEG-insulin group. The source of pegylated insulin for this study was 5KPEG insulin from Example 2. Doses were administered intraveneously.

-   -   Number/Sex of Animals    -   Day 1: IM/Group for the placebo Group 1    -   2M/Group for Group 2; 4M/Group for Groups 3-5

The animals were fasted for 12-18 hours prior to the initiation of thestudy. Human insulin (Diosynth) was stored at −20° C. prior to use. 5KPEG Insulin (Example 2) was stored at −20° C. prior to use. Twodifferent solutions for administration were prepared:

Solutions for I.V. Administration

-   -   Non-PEGylated Human Insulin (1.0 mg/ml Stock): 1.0 ml of PBS was        added to 1.0 mg of insulin powder.    -   5K PEG Insulin: (1.0 mg/ml Human Insulin-concentration is based        upon insulin rather than on the conjugate): 6.0 ml of PBS was        added to 11.7 mg of 5K PEG Insulin powder.

The animals were anesthetized with inhaled isoflurane. The i.v. doses(300 μL/animal) were given through the FVC and then the catheter wastied off to eliminate cross contamination with the blood draws. Allblood samples were drawn through the JVC. Phosphate buffered saline(PBS) was administered to Group 1 as a 300 μL i.v. dose. Non-pegylatedhuman insulin was administered to Group 2 as a 20 μg/animal i.v. dose.The PEGylated human insulin formulation was administered to Group 3 as a20 μg/animal i.v. dose, Group 4 as a 40 μg/animal i.v. dose, and Group 5as a 30 μg/animal i.v. dose. Blood samples (˜500 μL) were collected fromthe JVC at predose (2 to 0.25 hours prior to dosing), 10, 15, 30, 60,120, and 180 minutes postdose. A small amount of blood was placed on aglucose test strip for determination of blood glucose by the GlucometerElite glucose monitor (Bayer Corp., Elkart, Ind.). The remainder of thesample was placed into serum separator tubes and placed into thecentrifuge to separate the blood. The serum was then decanted into aseparate tube and analyzed by radioimmunoassay (RIA). Means and standarddeviations (SD) were calculated using Microsoft® Excel 2000.

TABLE 4 Summary of In-Vivo Experiments The following are the actualdoses administered and actual animal numbers per group that were used.The study was completed in one day. Total Daily Dose of Number of GroupRoute of Insulin Animals/ Number Composition Administration (□g/animal)Gender 1 Placebo i.v.  0 1M 2 Non-PEG Insulin i.v. 20 2M 3 PEG Insulini.v. 20 4M 4 PEG Insulin i.v. 40 4M 5 PEG Insulin i.v. 30 4M

The results demonstrate that the 5K PEG insulin composition possessesbioactivity, i.e., the insulin molecule remains active upon modificationwith polyethylene glycol, as can be seen by its ability to lower bloodglucose. Mean serum insulin concentrations following i.v. administrationof pegylated insulin were dose dependent; a dose dependent decrease inglucose levels was also observed. The results are summarized in FIGS. 2and 3. FIG. 2 is a plot of mean serum insulin concentrations followingi.v. administration of illustrative compositions of pegylated versusnon-pegylated insulin; FIG. 3 is a plot of blood glucose concentrationsfollowing i.v. administration of the compositions described above.

Example 8 Administration of 5K PEG Insulin to the Lung (P-2001-017)

An exemplary pegylated insulin, 5K PEG insulin, was administered to ratsvia intratracheal administration to determine (i) whether its activitywas maintained upon administration to the lung, and (ii) its impact, ifany, on serum insulin and blood glucose concentrations when delivereddirectly to the lung.

Stock Solutions

Non-PEGylated Insulin: 1 ml of PBS was added to 1.0 mg of insulin powderto prepare a 1 mg/ml stock solution. The stock solution of insulin(control) was prepared on the study initiation day.

5K PEG Insulin: 4.0 ml of PBS was added to 7.8 mg of 5K PEG Insulinpowder to prepare a 1 mg/ml (based on insulin) stock solution.

Dosing Solutions

40 μg/animal of Insulin: Within 2 hours of dosing, 667 μl of the insulinstock solution was added to 4.33 ml of PBS.

150 μg/animal of Insulin B-1: Within 2 hours of dosing, 2.5 ml of the 5KPEG Insulin stock solution was added to 2.5 ml of PBS.

Intratracheal Instillation

The rats were lightly anesthetized using inhaled 3.0-5.0% Isoflurane(Abbott Laboratories) mixed with oxygen for approximately 5 minutes in aplexiglass anesthesia chamber. Administration was accomplished byinsertion of a gavage needle (Popper & Sons Inc.; 18×3″ W2-¼ mm ball,New Hyde Park, N.Y. 11040) fitted into a 1 mL syringe into the mouth ofthe rat down the trachea to just above the main carina. When insertingthe gavage needle into the trachea, proper insertion was detected byfeeling for the roughness of the cartilage rings under the skin of thethroat using the ball of the gavage needle. Doses were administered intothe lungs utilizing this method, and followed by removal of the gavageneedle.

Fourteen (N=7/Group) fasted male rats (Hilltop Lab Animals, Scottsdale,Pa. (300-350 g)) with indwelling jugular vein catheters (JVC) were usedfor this study. Non-pegylated human insulin was administered to Group 1as a 40□g/300 □L i.t. dose. The PEGylated human insulin formulation wasadministered to Group 2 as a 150 □g/300 □L i.t. dose. Blood samples(˜500 □L) were collected at predose (2 to 0.25 hours prior to dosing),15, 30, 60, 120, 240, 360, 480, and 720 minutes postdose. A small amountof blood was placed on a glucose test strip for determination of bloodglucose in the Glucometer Elite glucose monitor (Bayer Corp., Elkart,Ind.). The remainder of the sample was placed into serum separator tubesand analyzed by radioimmunoassay. Means and standard deviations (SD)were calculated using Microsoft□ Excel 2000. Animal 2-3 was dropped fromthe study due to a clogged catheter.

TABLE 5 Summary of In-Vivo Experiments in Rats The following are theactual doses administered and actual animal numbers per group that wereused. Total Route of Number of Daily Dose No of Type of Adminis-Animals/ of Insulin Dosing Group No. Insulin tration Gender (μg/animal)Days 1 Insulin I.T. 7M  40 1 2 5K PEG I.T. 7M 150 1 Insulin

TABLE 6 In-Vivo Dose Levels Total Daily Dose Dose Concentration of GroupType of of Insulin Volume Dosing No. Insulin (μg/animal) (μl) Solution(μg/ml) 1 Insulin  40 300 133.33 2 5K PEG 150 300 500 Insulin

Mean serum concentrations and mean blood concentrations of insulin and5K PEG Insulin following intratracheal administration were plotted andare shown in FIG. 4 and in FIG. 5 respectively. The results demonstratethat the pegylated insulin compositions of the invention possessactivity upon delivery to and residence within the lung. Thepharmacokinetic data further demonstrate that pegylated insulin not onlypasses through the lung into the circulation, but does so whilstmaintaining activity, as evidenced by detectable serum insulin levelscorresponding to non-endogenous insulin. Due to the blood levels ofinsulin observed within about 1 hour following intratrachealadministration, it appears that pegylated insulin is not substantiallyheld up within the lungs and crosses the lungs into the bloodstreamshortly after administration. The results further indicate thatpegylated insulin, when administered to the lung, is effective inlowering blood glucose. However, in the present example, pegylatedinsulin appears less effective than non-pegylated insulin at the dosesadministered in lowering blood glucose. Both the pharmacokinetic andpharmacodynamic response curves for intratracheally administeredpegylated insulin somewhat resemble non-pegylated insulin, althoughbased upon the profiles in FIG. 4, PEG-insulin appears to be longeracting than non-pegylated insulin. Further optimization of the dosingamounts and particular polyethylene glycol modifiers may be readilyachieved by one of skill in the art, based upon the guidance presentedherein and depending upon the dosing requirements, intended patientpopulation, condition to be treated, and the like, of a particularchemically modified insulin product.

Example 9 Administration of 750-1 PEG Insulin to the Lung (P-2001-025)

A representative pegylated insulin, 750-1-PEG Insulin, was administeredto rats by intratracheal administration. The study was conducted, inpart, to explore the effects of a composition of insulin covalentlyattached to one or more polyethylene glycol chains having an approximatemolecular weight of 1,000 Daltons or less, when administered to thelung.

The in-vivo rat study was conducted essentially as described in Example8 above. The precise dosing regimen followed and doses administered aresummarized in the tables below.

TABLE 7 Total Route of Number of Daily Dose No of Type of Adminis-Animals/ of Insulin Dosing Group No. Insulin tration Gender (μg/animal)Days 1 Insulin I.T. 2M  80 1 2 750-1 I.T. 4M 100 1 PEG Insulin 3 750-1I.T. 4M 300 1 PEG Insulin 4 750-1 I.T. 4M 500 1 PEG Insulin

TABLE 8 Total Daily Dose Dose Concentration of Group Type of of InsulinVolume Dosing No. Insulin (μg/animal) (μl) Solution (μg/ml) 1 Insulin 80 300 266.67 2 750-1 100 300 333.33 PEG Insulin 3 750-1 300 3001000.00 PEG Insulin 4 750-1 500 300 1666.67 PEG Insulin

Serum insulin and blood glucose concentrations of insulin and 750-1 PEGInsulin following intratracheal administration in rats were plotted andare shown in FIG. 6 and FIG.7 respectively. When looking at the plot ofmean serum insulin concentrations in FIG. 6, native or non-pegylatedinsulin reached its maximum serum concentration at approximately 15minutes, while the pegylated insulin compositions reached maximum serumconcentrations at 6 hours (100 μg/animal) and 8 hours (300 μg/animal),demonstrating the long-acting nature of these compositions whenadministered to the lung by inhalation. As can be seen in FIG. 6,unmodified insulin returned to baseline at approximately 6 hours postadministration while the insulin levels for pegylated insulin at 6 hourswere significantly above baseline (from about 3 to 7 times or greaterthe baseline value). Moreover, intratracheal administration of pegylatedinsulin resulted in sustained levels of systemic insulin that had notreturned to baseline even at 12 hours post administration. In fact,insulin levels for pegylated insulin were over three times the baselinevalue (i.e., the value for non-modified insulin) at both 8 and 12 hours.A plot of these results is shown in FIG. 6.

In sum, when administered to the lung, 750-1 PEG insulin resulted inincreased systemic insulin levels when compared to unmodified insulin.Moreover, systemic insulin levels for the pegylated insulin group werestill significantly above baseline even at 12 hours. That is to say,elevated insulin levels were sustained for the pegylated insulin groupfor at least twice as long as for unmodified insulin. This data furtherdemonstrates that pegylated insulin crosses the lungs, is bioactive, andprovides prolonged systemic levels of insulin when compared tounmodified insulin.

A plot of mean blood glucose concentrations following intratrachealadministration of non-pegylated versus 750-1 pegylated insulin isprovided in FIG. 7. Blood glucose response levels correlated nicely withserum insulin levels for the pegylated insulin group. (That is to say,at elevated levels of serum insulin, a corresponding supression/loweringof blood glucose was also observed). In looking at FIG. 7, it can beseen that the pegylated insulin compositions of the invention, whenadministered orally to the lung, exhibit a rapid onset of actioncomparable to native insulin rather than a delayed onset of actiontypical of many sustained release formulations. That is to say,suppression of glucose occurs shortly after administration.Additionally, while native or non-pegylated insulin reaches maximumglucose lowering in about 2 hours, the time to reach maximum glucoselowering for pegylated insulin was extended to at least 4 hours, 6 hoursand 8 hours for the 500 μg, 100 μg, and 300 μg doses, respectively. So,the time to reach maximum blood glucose lowering for pegylated insulin,when administered to the lung, was prolonged 2 to 4 times over that ofnon-pegylated insulin. Overall, the 750-1 PEG insulin glucosesuppression was significantly increased over the 12 hour period whencompared with unmodified insulin. At 8 hours, glucose levels hadessentially returned to normal for unmodified insulin, while glucoselevels for the PEG insulin group were from 1.3 to 3 times lower than forunmodified insulin. Glucose levels for the pegylated insulin group hadnot returned to even at 12 hours, further indicating prolonged glucosesuppression for the lly derivatized insulin compositions of the presentinvention.

Example 10 Administration of 750-1 PEG Insulin to the Lung (P-2002-001)

In a study similar to Example 9 above, 750-1-PEG insulin wasadministered to intratracheal administration at doses lower than thoseemployed in Example 9.

The in-vivo intratracheal rat study was conducted essentially asdescribed in Example 8 above. The precise dosing regimen and dosesadministered are summarized in the tables below.

TABLE 9 Total Route of Number of Daily Dose No of Type of Adminis-Animals/ of Insulin Dosing Group No. Insulin tration Gender (μg/animal)Days 1 Insulin i.t. 5M 80 1 2 750-1 i.t. 5M 80 1 PEG Insulin 3 750-1 i.t5M 160 1 PEG Insulin

TABLE 10 Total Daily Dose Dose Concentration of Group Type of of InsulinVolume Dosing No. Insulin (μg/animal) (μl) Solution (μg/ml) 1 Insulin 80 300 266.7 2 750PEG  80 300 266.7 Insulin-1 3 750PEG 160 300 533.3Insulin-1

Serum insulin and blood glucose concentrations of unmodified insulin and750-1 PEG insulin following intratracheal administration in rats wereplotted and the results are shown in FIG. 8 and in FIG. 9, respectively.When looking at the plot of mean serum insulin concentrations in FIG. 8,native or non-pegylated insulin reached its maximum serum concentrationat approximately 15 minutes, while the pegylated insulin compositionsreached maximum serum concentrations at 2 hours (80 μg/animal) and 6hours (160 μg/animal). That is to say, the time to reach maximum serumlevels of insulin for peg-modified insulin was extended 8 to 24 timesover native or non-pegylated insulin when administered to the systemiccirculation via the lung. As can be seen in FIG. 8, unmodified insulinreturned to baseline at approximately 12 hours post administration,while insulin levels for the PEG-insulin group ranged from 2.5 to 3.5times the baseline value at the same 12 hour time point. Insulin levelsfor the pegylated insulin group did not return to baseline until around25 hours, meaning that it took twice as long for the pegylated insulingroup to return to baseline when compared to unmodified insulin.Systemic insulin levels were sustained for the pegylated insulin groupfor a duration of time about two-fold or twice (25 hours versus 12hours) that of unmodified insulin. At time points up to about 6 hours,the insulin levels for the two pegylated insulin groups roughlycorresponded to the doses administered (that is to say, insulinconcentrations for the 160 μg/animal group were approximately twicethose of the 80 μg/animal group).

A plot of mean blood glucose concentrations following intratrachealadministration of non-pegylated versus 750-1 pegylated insulin isprovided in FIG. 9. At 25 hours post administration, glucose suppressionfor both pegylated insulin groups had still not returned to baseline incontrast to unmodified insulin. Similar to the results from Example 9,the overall profiles for pegylated insulin demonstrate prolonged glucosesuppression extending beyond 25 hours. At 8 hours, glucose levels hadreturned to nearly normal for unmodified insulin while glucose levelsfor the PEG insulin groups were about 1.5 times lower than forunmodified insulin. These results further demonstrate that modifyinginsulin with one or more polyethylene glycol moieties results in goodbioavilability across the lungs and prolonged systemic insulin levels aswell as prolonged glucose suppression.

Example 11 Administration of 750-2 PEG Insulin to the Lung (P-2002-003)

A representative pegylated insulin composition, 750-2-PEG insulin, wasadministered to rats by intratracheal administration. This study wasconducted to further explore the effect of various doses of pegylatedversus non-pegylated insulin when administered directly to the lungs.Animals were dosed at 80 μg insulin per animal for both pegylated andnon-pegylated forms of insulin. The in-vivo rat study was conductedessentially as described in Example 8 above. The precise dosing regimenfollowed and doses administered are summarized in the tables below.

TABLE 11 Total Route of Number of Daily Dose No of Type of Adminis-Animals/ of Insulin Dosing Group No. Insulin tration Gender (μg/animal)Days 1 Insulin IT 7M 80 1 2 750PEG-2 IT 7M 80 1 Insulin

TABLE 12 Total Daily Dose Dose Concentration of Group Type of of InsulinVolume Dosing No. Insulin (μg/animal) (μl) Solution (μg/ml) 1 Insulin 80300 266.7 2 750PEG-2 80 300 266.7 Insulin

A plot of mean serum insulin concentrations following intratrachealinstillation of both non-pegylated and 750-1 PEG insulin at a dose of 80μg/animal is shown in FIG. 10. A plot of mean blood glucoseconcentrations following intratracheal instillation of bothnon-pegylated and 750-1 PEG insulin at a dose of 80 μg/animal is shownin FIG. 11. Results similar to those in Examples 9 and 10 were obtained.

A tabulation of pharmacokinetic parameters from Examples 10 and 11 isprovided below. Bioavailability is absolute bioavailability (i.e.,compared to intraveneously administered insulin).

TABLE 13 Mean Serum Insulin Pharmacokinetics AUC Dose μU* Abso- Type ofμg/ C_(Max) T_(Max) min/ lute Example Insulin Route animal μU/ml min mlBA  9 (P- Insulin IT 80 56 15 12878 2001- 25)  9 750-1 IT 100 64 36827954 PEG  9 750-1 IT 300 160 188 50691 PEG  9 750-1 IT 500 3474 184255881 PEG 10 (P- Insulin IT 80 132 15 28167 2002- 001) 10 750-1 IT 8056 210 36818 PEG 10 750-1 IT 160 117 78 60713 PEG IV Ref. Insulin IV 203057 5 44388 P-2002- 002 IV Ref. 750-2 IV 20 2638 7 63190 PEG IV Ref.750-2 IV 30 3510 5 62746 PEG 11 (P- insulin IT 80 89 24 22203 12.5 2002-003) 11 750-2 IT 80 164 73 57639 32** PEG 32*** *outlier was not removedfrom data set. **relative to IV 20 μg/animal dose. Value with outlierremoved was 22% ***relative to IV 30 μg/animal dose. Value with outlierremoved was unchanged.

Absolute bioavailability was calculated as follows:$\frac{({AUC})}{\left( {AUC}_{{IV}\quad{ins}} \right)}\frac{\left( {Dose}_{{IV}\quad{ins}} \right)}{({Dose})}$

Example 12 Administration of 2K PEG Insulin to the Lung (P-2002-010)

Another exemplary pegylated insulin composition, 2K PEG insulin, wasadministered to rats by intratracheal administration. The 2K PEG insulinused for this study was prepared as described in Example 3. Animals weredosed at 80 μg insulin per animal for non-pegylated insulin. Animalswere dosed at 300 μg insulin per animal, 600 μg insulin per animal, 900μg insulin per animal, and 1200 μg insulin per animal for 2K PEGinsulin. The in-vivo rat study was conducted essentially as described inExample 8 above. The precise dosing regimen followed and dosesadministered are summarized in the tables below.

TABLE 14 Total Route of Number of Daily Dose No of Type of Adminis-Animals/ of Insulin Dosing Group No. Insulin tration Gender (μg/animal)Days 1 Insulin i.t. 3 80 1 2 PEG2K-1 i.t. 3 600 1 Insulin 3 PEG2K-1 i.t.3 80 1 Insulin 4 PEG2K-1 i.t. 3 160 1 Insulin 5 PEG2K-1 i.t. 3 300 1Insulin 6 PEG2K-1 i.t. 3 900 1 Insulin 7 PEG2K-1 i.t. 3 1200 1 Insulin

TABLE 15 Total Daily Dose Dose Concentration of Group Type of of InsulinVolume Dosing No. Insulin (μg/animal) (μl) Solution (mg/ml) 1 Insulin 80300 0.267 2 PEG2K-1 600 300 2.0 Insulin 3 PEG2K-1 80 300 0.267 Insulin 4PEG2K-1 160 300 0.533 Insulin 5 PEG2K-1 300 300 1.0 Insulin 6 PEG2K-1900 300 3.0 Insulin 7 PEG2K-1 1200 300 4.0 Insulin

A plot of mean blood glucose concentrations following intratrachealadministration is shown in FIG. 12. Good dose response was observed forthe pegylated insulin compositions when administered to the lung (i.e.,higher doses of 2K PEG insulin resulted in a greater decrease in bloodglucose concentration). Although the time point in the curve at whichmaximum glucose suppression was achieved appears to be about 3 hours forboth the pegylated and non-pegylated compositions, the profiles for thepegylated versus non-pegylated insulin differ significantly with respectto duration of glucose suppression. In particular, for time points pastabout 6 hours, at the three higher 2K pegylated insulin doses (600 μg,900 μg, and 1200 μg per animal), glucose levels were suppressedsignificantly below those of non-pegylated insulin. These resultsfurther demonstrate that a prolonged systemic effect can be achieved byadministration of pegylated insulin to the lung.

Example 13 Evaluation of Serum Glucose and Insulin ConcentrationsFollowing Intravenous Administration of 2K PEG Insulin in Rats(P-2002-009)

This study was conducted to further explore the activity of insulin inan exemplary 2K PEG insulin composition, and to determine theintravenous (i.v.) dose of pegylated human insulin (PEG2K-1) effectiveto lower blood glucose to a concentration of about 30-40 mg/dL.

A protocol similar to that described in Example 7 was conducted usingthe compositions, animal groups, and doses summarized in the tablesbelow.

TABLE 16 Total Route of Number of Daily Dose No of Type of Adminis-Animals/ of Insulin Dosing Group No. Insulin tration Gender (μg/animal)Days 1 Insulin i.v. 2M 20 1 2 PEG2K-1 i.v. 2M 20 1 Insulin 3 PEG2K-1i.v. 2M 30 1 Insulin 4 PEG2K-1 i.v. 2M 40 1 Insulin 5 PEG2K-1 i.v. 2M 801 Insulin 6 PEG2K-1 i.v. 2M 160 1 Insulin

TABLE 17 Control/ Total Daily Dose Dose Concentration Test of InsulinVolume of Dosing Group No. Article (μg/animal) (μl) Solution (□g/ml) 1Insulin 20 300 67 2 PEG2 20 300 67 K-1 Insulin 3 PEG2 30 300 100 K-1Insulin 4 PEG2 40 300 133 K-1 Insulin 5 PEG2 80 300 267 K-1 Insulin 6PEG2 160 300 533 K-1 Insulin

A plot of mean serum insulin concentrations following intravenousadministration of non-pegylated and 2K PEG insulin at doses of 20μg/animal (non-pegylated insulin) and 20, 30 and 40 μg/animal (2K PEGinsulin) is shown in FIG. 13. A plot of mean blood glucoseconcentrations following intravenous administration of non-pegylated and2K PEG insulin at the doses described above is shown in FIG. 14.

1. A dry powder composition for pulmonary administration, saidcomposition comprising a conjugate of insulin covalently coupled to oneor more molecules of polyethylene glycol, wherein said powder (i) ischaracterized by having an emitted dose value of at least about 50% and(ii) when administered to a subject by inhalation, sustains elevatedblood levels of insulin in said subject for at least about 6 hours postadministration.
 2. The composition of claim 1, wherein said polymer isabsent a fatty acid moiety.
 3. The composition of claim 1, characterizedby an absolute pulmonary bioavailability that is greater than that ofnative insulin.
 4. The composition of claim 3, characterized by anabsolute pulmonary bioavailability that is at least twice that of nativeinsulin.
 5. The composition of claim 1, characterized by an absolutepulmonary bioavailability greater than 15%.
 6. The composition of claim5, characterized by an absolute pulmonary bioavailability greater than30%.
 7. The composition of claim 1, which when administered to the lung,is characterized by a Tmax that is at least three times that of nativeinsulin.
 8. The composition of claim 7 which when administered to thelung, is characterized by a Tmax that is at least five tints that ofnative insulin.
 9. The composition of claim 1, wherein said polyethyleneglycol is end-capped.
 10. The composition of claim 9, wherein saidpolyethylene glycol is end-capped with an alkoxy group.
 11. Thecomposition of claim 1, wherein said polyethylene glycol is selectedfrom the group consisting of linear polyethylene glycol, branchedpolyethylene glycol, forked polyethylene glycol, and dumbbellpolyethylene glycol.
 12. The composition of claim 11, wherein saidpolyethylene glycol comprises a biodegradable linkage.
 13. Thecomposition of claim 11, wherein said polyethylene glycol comprises anumber of (OCH₂CH₂) subunits selected from the group consisting of fromabout 2 to 300 subunits, from about 4 to 200 subunits, and from about 10to 100 subunits.
 14. The composition of claim 11, wherein saidpolyethylene glycol has a nominal avenge molecular weight from about 200to about 4000 daltons.
 15. The composition of claim 11, wherein saidpolyethylene glycol is linear.
 16. The composition of claim 14, whereinsaid polyethylene glycol has a nominal average molecular weight selectedfrom the group consisting of 200, 300, 400, 500, 600, 750, 1000, 1500,2000, 2500, 3000, 3500, and 4000 daltons.
 17. The composition of claim14, wherein said polyethylene glycol has a nominal average molecularweight from about 200 to about 2,000 daltons.
 18. The composition ofclaim 14, wherein said polyethylene glycol has a nominal averagemolecular weight from about 200 to about 1,000 daltons.
 19. Thecomposition of claim 1, wherein said insulin is native insulin.
 20. Thecomposition of claim 1, wherein said conjugate has a purity of greaterthan 90%.
 21. The composition of claim 1, wherein said insulin iscovalently coupled to polyethylene glycol at one or more of its aminosites.
 22. The composition of claim 21, wherein at least about 75% ofthe B-1Phe sites on Insulin are covalently coupled to polyethyleneglycol.
 23. The composition of claim 22, wherein at least about 90% ofthe B-1Phe sites on insulin are covalently coupled to polyethyleneglycol.
 24. The composition of claim 21, comprising a mixture ofmono-PEG substituted and di-PEG substituted and di-PEG substitutedconjugates of insulin.
 25. The composition of claim 24, furthercomprising a tri-PEG substituted conjugate of insulation.
 26. Thecomposition of claim 1, wherein said insulin is covalently coupled topolyethylene glycol via a linking moiety positioned at a terminus ofsaid polyethylene glycol.
 27. The composition of claim 1, wherein saidpolyethylene glycol, prior to coupling with insulin, possesses anactivated linking moiety at one terminus suitable for covalent couplingwith insulin.
 28. The composition of claim 27, wherein said activatedlinking moiety is suitable for coupling with reactive insulin aminogroups.
 29. The composition of claim 28, wherein said activated linkingmoiety comprises a reactive functional group selected from the groupconsisting of N-hydroxysuccinimide active esters, active carbonates,aldehydes, and acetals.
 30. The composition of claim 26, wherein insulinis covalently coupled to polyethylene glycol via an amide linkage. 31.The composition of claim 1 in aerosolized form.
 32. The composition ofclaim 1 in liquid or dry form.
 33. The composition of claim 1 furthercomprising a pharmaceutically acceptable excipient.
 34. The compositionof claim 1 in spray-dried form.
 35. The composition of claim 1, having aT_(g) greater than 50° C.
 36. The composition of claim 1, comprisingfrom 2% to 95% by weight PEG-insulin conjugate.
 37. The composition ofclaim 33, wherein said excipient is selected from the group consistingof carbohydrates, amino acids, dipeptides, tripeptides, and buffers. 38.The composition of claim 37, wherein said excipient is a di- ortripeptide containing two or more leucyl residues.
 39. The compositionof claim 1 having an MMD of less than 10 μm.
 40. The composition ofclaim 1, having an MMD of less than 4 μm.
 41. The composition of claim1, having an MMAD of less than 5 μm.
 42. The composition of claim 1,having an MMAD of less than 3.5 μm.
 43. The composition of claim 1,having a moisture content of less than 10% by weight.
 44. Thecomposition of claim 1, in a dosage receptacle.